Lactacystin analogs

ABSTRACT

Compounds related to lactacystin and lactacystin Beta-lactone, pharmaceutical compositions containing the compounds, and methods of use.

This Application is a 371 of PCT/US96/05072 filed Apr. 12, 1996 and aCIP of 08/421,583, filed Apr. 12, 1995.

BACKGROUND OF THE INVENTION

The invention relates generally to lactacystin and analogues thereof.

Eukaryotic cells contain multiple proteolytic systems, includinglysosomal proteases, calpains, ATP-ubiquitin-proteasome dependentpathway, and an ATP-independent nonlysosomal process. The major neutralproteolytic activity in the cytosol and nucleus is the proteasome, a 20S(700 kDa) particle with multiple peptidase activities. The 20S complexis the proteolytic core of a 26S (1500 kDa) complex that degrades orprocesses ubiquitin-conjugated proteins. Ubquitination marks a proteinfor hydrolysis by the 26S proteasome complex. Many abnormal orshort-lived normal polypeptides are degraded by theubiquitin-proteasome-dependent pathway. Abnormal peptides includeoxidant-damaged proteins (e.g., those having oxidized disulfide bonds),products of premature translational termination (e.g., those havingexposed hydrophobic groups which are recognized by the proteasome), andstress-induced denatured or damaged proteins (where stress is inducedby, e.g., changes in pH or temperature, or exposure to metals). Inaddition, some proteins, such as casein, do not required ubquitinationto be hydrolyzed by the proteasome.

The proteasome has chymotryptic, tryiptic, and peptidyl-glutamyl peptidehydrolizing activities, i.e., the proteasome can cleave peptides on thecarboxyl side of hydrophobic, basic, and acidic residues, respectively.

SUMMARY OF THE INVENTION

The invention relates to novel compounds structurally related tolactacystin and lactacystin β-lactone. The invention also relates topharmaceutical compositions including lactacystin and lactacystinanalogs.

One aspect of the invention is a pharmaceutical composition containing acompound having the formula

wherein Z¹ is O, S, SO₂, NH, or NR_(a), R_(a) being C₁₋₆ alkyl; X¹ is O,S, CH₂, two singly bonded H, CH(R_(b)) in the E or Z configuration, orC(R_(b))(R_(c)) in the E or Z configuration, each of R_(b) and R_(c),independently, being C₁₋₆ alkyl, C₆₋₁₂ aryl, C₃₋₈ cycloalkyl, C₃₋₈heteroaryl, C₃₋₈ heterocyclic radical, or halogen, X¹ being two singlybonded H when Z¹ is SO₂; Z² is O, S, NH, NR_(d), CHR¹, or CHOR¹ in the(R) or (S) configuration, wherein R_(d) is C₁₋₆ alkyl and R¹ is H,halogen, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl,NR_(d)R_(e) (except where Z² is CHOR¹), or the side chain of anynaturally occurring α-amino acid, or R¹ and R² taken together are abivalent moiety, provided that when R¹ and R² are taken together, Z¹ isNH or NR_(a) and Z² is CHR¹; R_(e) being H, C₁₋₆ alkyl, C₁₋₆ haloalkyl,C₂₋₆ alkenyl, or C₂₋₆ alkynyl, and the bivalent moiety forming a C₃₋₈cycloalkyl, C₃₋₈ heteroaryl, C₃₋₈ heterocyclic radical, or C₆₋₁₂ aryl,where the H in CHR¹ is deleted when R₁ and R₂ taken together form a C₃₋₈heteroaryl or C₆₋₁₂ aryl; R² is C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆alkenyl, azido, C₂₋₆ alkynyl, halogen, OR_(f), SR_(f), NR_(f)R_(g),—ONR_(f)R_(g), —NR_(g)(OR_(f)) or NR_(g)(SR_(f)) (each of R_(f) andR_(g), independently, being H, C₁₋₆, alkyl, C₁₋₆ haloalkyl, C₂₋₆alkenyl, or C₂₋₆ alkynyl), or R¹ and R² taken together are a bivalentmoiety, the bivalent moiety forming a C₃₋₈ cycloalkyl, C₃₋₈ heteroaryl,C₃₋₈ heterocyclic radical, or C₆₋₁₂ aryl, where the H in CHR¹ is deletedwhen R₁ and R₂ taken together form a C₃₋₈ heteroaryl or C₆₋₁₂ aryl; A¹is H, the side chain of any naturally occurring α-amino acid, or is ofthe following formula,

—(CH₂)_(m)—Y—(CH₂)_(n)—R³X³

wherein Y is O, S, C═O, C═S, —(CH═CH)—, vinylidene, —C═NOR_(h),—C═NNR_(i)R_(i′), sulfonyl, methylene, CHX⁴ in the (R) or (S)configuration, or deleted, X⁴ being halogen, methyl, halomethyl, OR_(h),SR_(h), NR_(i)R_(i′), —NR_(i)(OR_(h)), or —NR_(i)(NR_(i)R_(i′)), whereinR_(h) is selected from H, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, C₂₋₆alkynyl, C₁₋₁₀ acyl, C₁₋₆ alkylsulfonyl, and C₆₋₁₀ arylsulfonyl; andeach of R_(i) and R_(i′), independently is selected from H, C₁₋₆ alkyl,C₁₋₆ haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, and C₁₋₁₀ acyl; m is 0, 1,2, or 3, and n is 0, 1, 2, or 3; and R³ is straight chain or branchedC₁₋₈ alkylidene, straight chain or branched C₁₋₈ alkylene, C₃₋₁₀cycloalkylidene, C₃₋₁₀ cycloalkylene, phenylene, C₆₋₁₄ arylalkylidene,C₆₋₁₄ arylalkylene, or deleted, and X³ is H, hydroxyl, thiol, carboxyl,amino, halogen, (C₁₋₆ alkyl)oxycarbonyl, (C₇₋₁₄ arylalkyl)oxycarbonyl,or C₆₋₁₄ aryl; or R³ and X³ taken together are the side chain of anynaturally occurring α-amino acid; and L^(O) is an organic moiety having1 to 25 carbon atoms, 0 to 10 heteroatoms, and 0 to 6 halogen atoms; anda pharmaceutically acceptable carrier.

A second aspect is a pharmaceutical composition comprising a compoundhaving the following formula

wherein Z¹ is O, S, SO₂, NH, or NR_(a), R_(a) being C₁₋₆ alkyl; X¹ is O,S, CH₂, two singly bonded H, CH(R_(b)) in the E or Z configuration, orC(R_(b))(R_(c)) in the E or Z configuration, each of R_(b) and R_(c),independently, being C₁₋₆ alkyl, C₆₋₁₂ aryl, C₃₋₈ cycloalkyl, C₃₋₈heteroaryl, C₃₋₈ heterocyclic radical, or halogen, provided that when Z¹is SO₂, X¹ is two singly bonded H; Z² is CHR¹ in the (R) or (S)configuration, R¹ being H, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl,C₂₋₆ alkynyl, hydroxyl, halogen, a side chain of a naturally occuringα-amino acid, OR_(d), SR_(d), or NR_(d)R_(e) (each of R_(d) and R_(e),independently, being H, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, orC₂₋₅ alkynyl); Z³ is O, S, NH, or NR_(j), wherein R_(j) is C₁₋₆ alkyl;X² is O or S; and A¹ is H, the side chain of any naturally occurringα-amino acid, or is of the following formula,

—(CH₂)_(m)—Y—(CH₂)_(n)—R³X³

wherein Y is O, S, C═O, C═S, —(CH═CH)—, vinylidene, —C═NOR_(h),—C═NNR_(i)R_(i′), sulfonyl, methylene, CHX⁴ in the (R) or (S)configuration, or deleted, X⁴ being halogen, methyl, halomethyl, OR_(h),SR_(h), NR_(i)R_(i′), —NR_(i)(OR_(h)), or —NR_(i)(NR_(i)R_(i′)), whereinR_(h) is selected from H, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, C₂₋₆alkynyl, C₁₋₁₀ acyl, C₁₋₆ alkylsulfonyl, and C₆₋₁₀ arylsulfonyl; andeach of R_(i) and R_(i′), independently is selected from H, C₁₋₆ alkyl,C₁₋₆ haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, and C₁₋₁₀ acyl; m is 0, 1,2, or 3, and n is 0, 1, 2, or 3; R³ is straight chain or branched C₁₋₈alkylidene, straight chain or branched C₁₋₈ alkylene, C₃₋₁₀cycloalkylidene, C₃₋₁₀ cycloalkylene, phenylene, C₆₋₁₄ arylalkylidene,C₆₋₁₄ arylalkylene, or deleted; and X³ is H, hydroxyl, thiol, carboxyl,amino, halogen, (C₁₋₆ alkyl)oxycarbonyl, (C₇₋₁₄ arylalkyl)oxycarbonyl,or C₆₋₁₄ aryl; or R³ and X³ taken together are the side chain of anynaturally occurring α-amino acid; and a pharmaceutically acceptablecarrier.

A third aspect is a pharmaceutical composition comprising a compoundhaving one of the following formulae

wherein Z¹ is NH or NR_(a), R_(a) being C₁₋₆ alkyl; X¹ is O, S, CH₂, twosingly bonded H, CH(R_(b)) in the E or Z configuration, or C(R_(b))(R_(c)) in the E or Z configuration, each of R_(b) and R_(c),independently, being C₁₋₆ alkyl, C₆₋₁₂ aryl, C₃₋₈ cycloalkyl, C₃₋₈heteroaryl, C₃₋₈ heterocyclic radical, or halogen; Z² is O, S, NH, orNR_(j), wherein R_(j) is C₁₋₆ alkyl; R¹ is in the (R) or (S)configuration, and is H, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, C₂₋₆alkynyl, hydroxyl, halogen, a side chain of a naturally occurringα-amino acid, OR_(d), SR_(d), or NR_(d)R_(e) (each of R_(d) and R_(e),independently, being H, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₆₋₁₂ aryl, C₃₋₈cycloalkyl, C₃₋₈ heteroaryl, C₃₋₈ heterocyclic radical, or halogen); X¹is O or S; and A¹ is H, the side chain of any naturally occurringα-amino acid, or is of the following formula,

—(CH₂)_(m)—Y—(CH₂)_(n)—R³X³

wherein Y is O, S, C═O, C═S, —(CH═CH)—, vinylidene, —C═NOR_(h),—C═NNR_(i)R_(i′), sulfonyl, methylene, CHX⁴ in the (R) or (S)configuration, or deleted, X⁴ being halogen, methyl, halomethyl, OR_(h),SR_(h), NR_(i)R_(i′), —NR_(i)(OR_(h)), or —NR_(i)(NR_(i)R_(i′)), whereinR_(h) is selected from H, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, andC₂₋₆ alkynyl, and each of R_(i) and R_(i′), independently is selectedfrom H, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, andC₁₋₁₀ acyl; m is 0, 1, 2, or 3, and n is 0, 1, 2, or 3; and R³ isstraight chain or branched C₁₋₈ alkylidene, straight chain or branchedC₁₋₈ alkylene, C₃₋₁₀ cycloalkylidene, C₃₋₁₀ cycloalkylene, phenylene,C₆₋₁₄ arylalkylidene, C₆₋₁₄ arylalkylene, or deleted, and X³ is H,hydroxyl, thiol, carboxyl, amino, halogen, (C₁₋₆ alkyl)oxycarbonyl,(C₇₋₁₄ arylalkyl)oxycarbonyl, or C₆₋₁₄ aryl; or R³ and X³ taken togetherare the side chain of any naturally occurring α-amino acid; and apharmaceutically acceptable carrier.

A fourth aspect is a pharmaceutical composition containing a compoundhaving the following formula

wherein Z¹ is O, S, NH or NR_(j), R_(j) being C₁₋₆ alkyl; X¹ is O or S;R¹ is in the (R) or (S) configuration, and is H, C₁₋₆ alkyl, C₁₋₆haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, hydroxyl, halogen, a side chainof a naturally occuring α-amino acid, OR_(d), SR_(d), or NR_(d)R_(e)(each of R_(d) and R_(e), independently, being H, C₁₋₆ alkyl, C₁₋₆haloalkyl, C₂₋₆ alkenyl, or C₂₋₅ alkynyl); R² is H, C₁₋₆ alkyl, C₁₋₆haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₆₋₁₂ aryl, C₃₋₈ heteroaryl, orhalogen; X² is O or S; and A¹ is H, the side chain of any naturallyoccurring α-amino acid, or is of the following formula,

—(CH₂)_(m)—Y—(CH₂)_(n)—R³X³

wherein Y is O, S, C═O, C═S, —(CH═CH)—, vinylidene, —C═NOR_(h),—C═NNR_(i)R_(i′), sulfonyl, methylene, CHX⁴ in the (R) or (s)configuration, or deleted, X⁴ being halogen, methyl, halomethyl, OR_(h),SR_(h), NR_(i)R_(i′), —NR_(i)(OR_(h)), or —NR_(i)(NR_(i)R_(i′)), whereinR_(h) is selected from H, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, andC₂₋₆ alkynyl, and each of R_(i) and R_(i), independently is selectedfrom H, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, andC₁₋₁₀ acyl; m is 0, 1, 2, or 3, and n is 0, 1, 2, or 3; and R³ isstraight chain or branched C₁₋₈ alkylidene, straight chain or branchedC₁₋₈ alkylene, C₃₋₁₀ cycloalkylidene, C₃₋₁₀ cycloalkylene, phenylene,C₆₋₁₄ arylalkylidene, C₆₋₁₄ arylalkylene, or deleted, and X³ is H,hydroxyl, thiol, carboxyl, amino, halogen, (C₁₋₆ alkyl)oxycarbonyl,(C₇₋₁₄ arylalkyl)-oxycarbonyl, or C₆₋₁₄ aryl; or R³ and X³ takentogether are the side chain of any naturally occurring α-amino acid; anda pharmaceutically acceptable carrier.

A fifth aspect is a pharmaceutical composition comprising a compoundhaving the following formula

wherein Z¹ is O, S, NH or NR_(j), R_(j) being C₁₋₆ alkyl; X¹ is O or S;R¹ is H, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl,hydroxyl, halogen, a side chain of a naturally occuring α-amino acid,OR_(d), SR_(d), or NR_(d)R_(e) (each of R_(d) and R_(e), independently,being C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₆₋₁₂ aryl, C₃₋₈ cycloalkyl, C₃₋₈heteroaryl, C₃₋₈ hetero-cyclic radical, or halogen); R² is H, C₁₋₆alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₆₋₁₂ aryl, C₃₋₈heteroaryl, or halogen; R_(a) is C₁₋₆ alkyl; and A¹ is H, the side chainof any naturally occurring α-amino acid, or is of the following formula,

—(CH₂)_(m)—Y—(CH₂)_(n)—R³X³

wherein Y is O, S, C═O, C═S, —(CH═CH)—, vinylidene, —C═NOR_(h),—C═NNR_(i)R_(i′), sulfonyl, methylene, CHX⁴ in the (R) or (S)configuration, or deleted, X⁴ being halogen, methyl, halomethyl, OR_(h),SR_(h), NR_(i)R_(i′), —NR_(i)(OR_(h)), or —NR_(i)(NR_(i)R_(i′)), whereinR_(h) is selected from H, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, andC₂₋₆ alkynyl, and each of R_(i) and R_(i′), independently is selectedfrom H, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, andC₁₋₁₀ acyl; m is 0, 1, 2, or 3, and n is 0, 1, 2, or 3; and R³ isstraight chain or branched C₁₋₈ alkylidene, straight chain or branchedC₁₋₈ alkylene, C₃₋₁₀ cycloalkylidene, C₃₋₁₀ cycloalkylene, phenylene,C₆₋₁₄ arylalkylidene, C₆₋₁₄ arylalkylene, or deleted, and X³ is H,hydroxyl, thiol, carboxyl, amino, halogen, (C₁₋₆ alkyl)oxycarbonyl,(C₇₋₁₄ arylalkyl)-oxycarbonyl, or C₆₋₁₄ aryl; or R³ and X³ takentogether are the side chain of any naturally occurring α-amino acid; anda pharmaceutically acceptable carrier.

A sixth aspect is a pharmaceutical composition containing a compoundhaving the formula

wherein X¹ is O, S, CH₂, two singly bonded H, CH(R_(b)) in the E or Zconfiguration, or C(R_(b))(R_(c)) in the Eor Z configuration, each ofR_(b) and R_(c), independently, being C₁₋₆ alkyl, C₆₋₁₂ aryl, C₃₋₈cycloalkyl, C₃₋₆ heteroaryl, C₃₋₈ heterocyclic radical, or halogen; Z¹is O, S, NH, or NR_(a), R_(a) being C₁₋₆ alkyl; R¹ is H, C₁₋₆ alkyl,C₁₋₆ haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, hydroxyl, halogen, a sidechain of any naturally occuring α-amino acid, OR_(d), SR_(d), orNR_(d)R_(e) (each of R_(d) and R_(e), independently, being H, C₁₋₆alkyl, C₁₋₆ halo-alkyl, C₆₋₁₂ aryl, C₃₋₈ cycloalkyl, C₃₋₈ heteroaryl,C₃₋₈ heterocyclic radical, or halogen); or R¹ and R² taken together area bivalent moiety which forms a C₃₋₈ cycloalkyl, C₃₋₈ heteroaryl, C₃₋₈heterocyclic radical, or C₆₋₁₂ aryl; R² is H, C₁₋₆ alkyl, C₁₋₆haloalkyl, C₂₋₆ alkenyl, azido, C₂₋₆ alkynyl, halogen, OR_(f), SR_(f),NR_(f)R_(g), —ONR_(f)R_(g), NR_(g)(OR_(f)), or NR_(g)(SR_(f)) (each ofR_(f) and R_(g), independently, being H, C₁₋₆, alkyl, C₁₋₆ haloalkyl,C₂₋₆ alkenyl, or C₂₋₆ alkynyl), or R¹ and R² taken together are abivalent moiety, the bivalent moiety forming a C₃₋₈ cycloalkyl, C₃₋₈heteroaryl, C₃₋₈ heterocyclic radical, or C₆₋₁₂ aryl; X² is O or S; andA¹ is in the (R) or (S) configuration, and each of A¹ and A² isindependently H, the side chain of any naturally occurring amino α-acid,or is of the following formula,

—(CH₂)_(m)—Y—(CH₂)_(n)—R³X³

wherein Y is O, S, C═O, C═S, —(CH═CH)—, vinylidene, —C═NOR_(h),—C═NNR_(i)R_(i′), sulfonyl, methylene, CHX⁴ in the (R) or (S)configuration, or deleted, X⁴ being halogen, methyl, halomethyl, OR_(h),SR_(h), NR_(i)R_(i′), —NR_(i)(OR_(h)), or —NR_(i)(NR_(i)R_(i′)), whereinR_(h) is selected from H, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, andC₂₋₆ alkynyl, and each of R_(i) and R_(i), independently is selectedfrom H, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, andC₁₋₁₀ acyl; m is 0, 1, 2, or 3, and n is 0, 1, 2, or 3; and R³ isstraight chain or branched C₁₋₈ alkylidene, straight chain or branchedC₁₋₈ alkylene, C₃₋₁₀ cycloalkylidene, C₃₋₁₀ cycloalkylene, phenylene,C₆₋₁₄ arylalkylidene, C₆₋₁₄ arylalkylene, or deleted, and X³ is H,hydroxyl, thiol, carboxyl, amino, halogen, (C₁₋₆ alkyl)oxycarbonyl,(C₇₋₁₄ arylalkyl)-oxycarbonyl, or C₆₋₁₄ aryl; or R³ and X³ takentogether are the side chain of any naturally occurring α-amino acid; anda pharmaceutically acceptable carrier.

A seventh aspect is a pharmaceutical composition containing a compoundhaving the following formula

wherein Z¹ is NH or NR_(a), NR_(a) being C₁₋₆ alkyl;

X¹ is O, S, CH₂, two singly bonded H, CH(R_(b)) in the E or Zconfiguration, or C(R_(b))(R_(c)) in the E or Z configuration, each ofR_(b) and R., independently, being C₁₋₆ alkyl, C₆₋₁₂ aryl, C₃₋₈cycloalkyl, C₃₋₈ heteroaryl, C₃₋₈ heterocyclic radical, or halogen;

Z² is O, S, NH, or NR_(j), R_(j) being C₁₋₆ alkyl;

R¹ is in the (R) or (S) configuration, and is H, C₁₋₆ alkyl, C₁₋₆haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, hydroxyl, halogen, a side chainof any naturally occuring amino acid, OR_(d), SR_(d), or NR_(d)R_(e)(each of R_(d) and R_(e), independently, being H, C₁₋₆ alkyl, C₁₋₆haloalkyl, C₆₋₁₂ aryl, C₃₋₈ cycloalkyl, C₃₋₈ heteroaryl, C₃₋₈heterocyclic radical, or halogen); or R¹ and R² taken together are abivalent moiety which forms a C₃₋₈ cycloalkyl, C₃₋₈ heteroaryl, C₃₋₈heterocyclic radical, or C₆₋₁₂ aryl; R² is H, C₁₋₆ alkyl, C₁₋₆haloalkyl, C₂₋₆ alkenyl, azido, C₂₋₆ alkynyl, halogen, OR_(f), SR_(f),NR_(f)R_(g), —ONR_(f)R_(g), —NR_(g)(OR_(f)), or —NR_(g)(SR_(f)) (each ofR_(f) and R_(g), independently, being H, C_(1-6,) alkyl, C₁₋₆ haloalkyl,C₂₋₆ alkenyl, or C₂₋₆ alkynyl), or R¹ and R² taken together are abivalent moiety, the bivalent moiety forming a C₃₋₈ cycloalkyl, C₃₋₈heteroaryl, C₃₋₈ heterocyclic radical, or C₆₋₁₂ aryl; X² is O or S; andeach of A¹ and A² is independently in the (R) or (S) configuration, andis independently H, the side chain of any naturally occurring α-aminoacid, or is of the following formula,

—(CH₂)_(m)—Y—(CH₂)_(n)—R³X³

wherein Y is O, S, C═O, C═S, —(CH═CH)—, vinylidene, —C═NOR_(h),—C═NNR_(i)R_(i′), sulfonyl, methylene, CHX⁴ in the (R) or (S)configuration, or deleted, X⁴ being halogen, methyl, halomethyl, OR_(h),SR_(h), NR_(i)R_(i′), —NR_(i)(OR_(h)), or —NR_(i)(NR_(i)R_(i′)), whereinR_(h) is selected from H, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, andC₂₋₆ alkynyl, and each of R_(i) and R_(i′), independently is selectedfrom H, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, andC₁₋₁₀ acyl; m is 0, 1, 2, or 3, and n is 0, 1, 2, or 3; and R³ isstraight chain or branched C₁₋₈ alkylidene, straight chain or branchedC₁₋₈ alkylene, C₃₋₁₀ cycloalkylidene, C₃₋₁₀ cycloalkylene, phenylene,C₆₋₁₄ arylalkylidene, C₆₋₁₄ arylalkylene, or deleted, and X³ is H,hydroxyl, thiol, carboxyl, amino, halogen, (C₁₋₆ alkyl)oxycarbonyl,(C₇₋₁₄ arylalkyl)-oxycarbonyl, or C₆₋₁₄ aryl; or R³ and X³ takentogether are the side chain of any naturally occurring α-amino acid; anda pharmaceutically acceptable carrier.

An eighth aspect is a pharmaceutical composition containing a compoundof the formula

wherein Z¹ is O, NH, or NR_(a), NR_(a) being C₁₋₆ alkyl; X¹ is O, S,CH₂, or two singly bonded H; each of A¹ and A² is independently H, theside chain of any naturally occurring α-amino acid, or is of thefollowing formula,

—(CH₂)_(m)—Y—(CH₂)_(n)—R³X³

wherein Y is O, S, C═O, C═S, —(CH═CH)—, vinylidene, —C═NOR_(h),—C═NNR_(i)R_(i′), sulfonyl, methylene, CHX⁴ in the (R) or (S)configuration, or deleted, X⁴ being halogen, methyl, halomethyl, OR_(h),SR_(h), NR_(i)R_(i′), —NR_(i)(OR_(h)), or —NR_(i)(NR_(i)R_(i′)), whereinR_(h) is selected from H, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, andC₂₋₆ alkynyl, and each of R_(i) and R_(i), independently is selectedfrom H, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, andC₁₋₁₀ acyl; m is 0, 1, 2, or 3, and n is 0, 1, 2, or 3; and R³ isstraight chain or branched C₁₋₈ alkylidene, straight chain or branchedC₁₋₈ alkylene, C₃₋₁₀ cycloalkylidene, C₃₋₁₀ cycloalkylene, phenylene,C₆₋₁₄ arylalkylidene, C₆₋₁₄ arylalkylene, or deleted, and X³ is H,hydroxyl, thiol, carboxyl, amino, halogen, (C₁₋₆ alkyl)oxycarbonyl,(C₇₋₁₄ arylalkyl)-oxycarbonyl, or C₆₋₁₄ aryl; or R³ and X³ takentogether are the side chain of any naturally occurring α-amino acid; andR¹² is H, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, or C₂₋₆ alkynyl; anda pharmaceutically acceptable carrier.

A ninth aspect is a pharmaceutical composition comprising a compoundhaving the formula

wherein Z¹ is NH, or NR_(a), NR_(a) being C₁₋₆ alkyl; each of X¹ and X²,independently, is O or S; each of A¹ and A² is independently in the (R)or (S) configuration, and is independently H, the side chain of anynaturally occurring amino acid, or is of the following formula,

—(CH₂)_(m)—Y—(CH₂)_(n)—R³X³

wherein Y is O, S, C═O, C═S, —(CH═CH)—, vinylidene, —C═NOR_(h),—C═NNR_(i)R_(i′), sulfonyl, methylene, CHX⁴ in the (R) or (s)configuration, or deleted, X⁴ being halogen, methyl, halomethyl, OR_(h),SR_(h), NR_(i)R_(i′), —NR_(i)(OR_(h)), or —NR_(i)(NR_(i)R_(i′)), whereinR_(h) is selected from H, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, andC₂₋₆ alkynyl, and each of R_(i) and R_(i′), independently is selectedfrom H, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, andC₁₋₁₀ acyl; m is 0, 1, 2, or 3, and n is 0, 1, 2, or 3; and R³ isstraight chain or branched C₁₋₈ alkylidene, straight chain or branchedC₁₋₈ alkylene, C₃₋₁₀ cycloalkylidene, C₃₋₁₀ cycloalkylene, phenylene,C₆₋₁₄ arylalkylidene, C₆₋₁₄ arylalkylene, or deleted, and X³ is H,hydroxyl, thiol, carboxyl, amino, halogen, (C₁₋₆ alkyl)oxycarbonyl,(C₇₋₁₄ arylalkyl)-oxycarbonyl, or C₆₋₁₄ aryl; or R³ and X³ takentogether are the side chain of any naturally occurring α-amino acid; anda pharmaceutically acceptable carrier.

Many of the compounds described above are novel compounds; the novelcompounds are also claimed. The invention also encompasses lactacystinanalogues that can be made by the synthetic routes described herein, andmethods of treating a subject having a condition mediated by proteinsprocessed by the proteasome by administering (e.g., orally) to a subjectan effective amount of a pharmaceutical composition disclosed herein.These methods include treatments for cancer, inflammation (e.g.,inflammatory responses associated with allergies, bone marrow or solidorgan transplantation, or disease states), psoriasis, and restenosis.

The compounds disclosed herein are highly selective for the proteasome,and do not inhibit other proteases such as trypsin, α-chymotrypsin,calpain I, calpain II, papain, and cathepsin B.

Other features or advantages of the present invention will be apparentfrom the following detailed description, and also from the appendingclaims.

DETAILED DESCRIPTION OF THE INVENTION

Terms

The term “naturally occurring amino acid” is meant to include the 20common α-amino acids (Gly, Ala, Val, Leu, Ile, Ser, Thr, Asp, Asn, Lys,Glu, Gln, Arg, His, Phe, Cys, Trp, Tyr, Met and Pro), and other aminoacids that are natural products, such as norleucine, ethylglycine,ornithine, methylbutenylmethylthreonine, and phenylglycine. Examples ofamino acid side chains include H (glycine), methyl (alanine),—(CH₂—(C═O)—NH₂ (asparagine), —CH₂—SH (cysteine), and —CH(OH)CH₃(threonine).

The term “inhibitor” is meant to describe a compound that blocks orreduces the activity of an enzyme (e.g. the proteasome, or the X/MB1subunit or α-chain of the 20S proteasome). An inhibitor may act withcompetitive, uncompetitive, or noncompetitive inhibition. An inhibitormay bind reversibly or irreversibly, and therefore the term includescompounds which are suicide substrates of an enzyme. An inhibitor maymodify one or more sites on or near the active site of the enzyme, or itmay cause a conformational change elsewhere on the enzyme. Thus, somecompounds disclosed herein (e.g., where L^(O) is an oxiranyl or aldehydegroup) react with the enzyme by bonding to the carbon atom correspondingto C₄ of lactacystin (e.g., resulting in a C₄ having a hydroxyl orthiol), while other compounds react with the enzyme to release a leavinggroup (e.g., L¹) corresponding to an acylation.

An alkyl group is a branched or unbranched hydrocarbon that may besubstituted or unsubstituted. Examples of branched alkyl groups includeisopropyl, sec-butyl, isobutyl, tert-butyl, sec-pentyl, isopentyl,tert-pentyl, isohexyl. Substituted alkyl groups may have one, two, threeor more substituents, which may be the same or different, each replacinga hydrogen atom. Substituents are halogen (e.g., F, Cl, Br, and I),hydroxyl, protected hydroxyl, amino, protected amino, carboxy, protectedcarboxy, cyano, methylsulfonylamino, alkoxy, acyloxy, nitro, and lowerhaloalkyl. Similarly, cycloalkyl, aryl, arylalkyl, alkylaryl,heteroaryl, and heterocyclic radical groups may be substituted with oneor more of the above substituting groups. Examples of cycloalkyl groupsare cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, andcyclooctyl. An aryl group is a C₆₋₂₀ aromatic ring, wherein the ring ismade of carbon atoms (e.g., C₆₋₁₄, C₆₋₁₀ aryl groups, wherein C₈ arylmay be an alkylaryl such as 2,3-dimethylphenyl or an arylalkyl such asphenylethyl, or an alkylarylalkyl such as o-methyl-benzyl). Examples ofhaloalkyl include fluoromethyl, dichloromethyl, trifluoromethyl,1,1-difluoroethyl, and 2,2-dibromoethyl.

A heterocyclic radical contains at least one ring structure whichcontains carbon atoms and at least one heteroatom (e.g., N, O, S, or P).A heteroaryl is an aromatic heterocyclic radical. Examples ofheterocyclic radicals and heteroaryl groups include: thiazolyl, thienyl,furyl, 1-isobenzofuranyl, 2H-chromen-3-yl, 2H-pyrrolyl, N-pyrrolyl,imidazolyl, pyrazolyl, isothiazolyl, isooxazolyl, pyridyl, pyrazinyl,pyrimidinyl, pyradazinyl, indolizinyl, isoindolyl, indolyl, indazolyl,purinyl, phthalazinyl, cinnolinyl, and pteridinyl. A heterocylic radicalmay be attached to another moiety via a carbon atom or a heteroatom ofthe heterocyclic radical.

A (C_(n) alkyl)oxycarbonyl group has the formula R—O—(C═O)—. (C₁₋₆alkyl)oxycarbonyl, therefore, includes methoxycarbonyl andhexyloxycarbonyl. A C₁₋₁₀ acyl group as used herein is of the formula—(C═O)—L₃ and contains 1 to 10 carbon atoms (e.g., C₁₋₆) and 1-5heteroatoms (at least one oxygen). Examples of such acyl groups includeformyl, acetyl, benzyloxycarbonyl, tert-butoxycarbonyl,trifluoroethyloxycarbonyl, thiobenzoyl, phenylamidocarbonyl, and4-nitrophenoxycarbonyl.

An alkylene is a bivalent radical derived from alkanes by removing twohydrogens from two different carbon atoms. Examples of alkylenes include—CH₂—CH(R)—CH₂ and 1,4-cyclohexylene. An alkylidene is a bivalentradical derived from alkenes by removing two hydrogens from the samecarbon atom, such as 1-propanyl-3-ylidene (═CH—CH₂—CH₂—).

An aromatic carbon atom, as used herein, is a carbon atom within anaromatic system such as benzene or naphthalene or a heteroaromaticsystem such as quinoline. Examples of nonaromatic carbon atoms includethe carbons atoms in R—(C═O)—R, —CH₂Cl, —CH₂— and R—(C═O)—O—R. Afragment formula weight is the combined atomic weight of the fragment ormoiety indicated. For example, the fragment formula weight of methyl is15 and the fragment formula weight of hydroxyl is 17.

A leaving group departs from a substrate with the air of electrons ofthe covalent bond between the leaving group and the substrate; preferredleaving groups stabilize those electrons via the presence of electronwithdrawing groups, aromaticity, resonance structures, or a combinationthereof. Examples include halide (I and Br are preferred), mesylate,trifluoromethanesulfonate, p-toluenesulfonate, p24-nitrobenzensulfonate,benzoate, p-nitrobenzoate, p-nitrobenzyl, and C₂₋₅ haloalkylcarbonyloxysuch as trifluoroacetate.

Numerous thiol-, amino-, hydroxy- and carboxy-protecting groups arewell-known to those in the art. In general, the species of protectinggroup is not critical, provided that it is stable to the conditions ofany subsequent reaction(s) on other positions of the compound and can beremoved at the appropriate point without adversely affecting theremainder of the molecule. In addition, a protecting group may besubstituted for another after substantive synthetic transformations arecomplete. Clearly, where a compound differs from a compound disclosedherein only in that one or more protecting groups of the disclosedcompound has been substituted with a different protecting group, thatcompound is within the invention. For example, where a compound differsfrom a compound disclosed herein only in that one or more hydrogens froma thiol-, amino-, or hydroxyl-moiety (or a hydrogen or hydroxyl from acarboxyl moiety) has been substituted with a protecting group to form acarboxyl or sulfonyl ester, that protected compound is within theinvention. Sulfonyl esters include alkylsulfonyls (e.g., methylsulfonylor mesyl) and arylsulfonyls (e.g., tosyl). Further examples andconditions are found in T. W. Greene, Protective Groups in organicChemistry, (1st ed., 1981; 2nd ed., 1991, T. W. Greene and P. G. M.Wuts).

The invention also encompasses isotopically-labelled counterparts ofcompounds disclosed herein. An isotopically-labelled compound of theinvention has one or more atoms replaced with an isotope having adetectable particle- or x-ray-emitting (radioactive) nucleus or amagnetogyric nucleus. Examples of such nuclei include ²H, ³H, ¹³C, ¹⁵N,¹⁹F, ²⁹Si, ³¹p, ³²p and ¹²⁵I. Isotopically-labelled compounds of theinvention are particularly useful as probes or research tools forspectrometric analyses, radioimmunoassays, binding assays based on γ- orβ-scintillation, fluorography, autoradiography, and kinetic studies suchas inhibition studies or determination of primary and secondary isotopeeffects.

The following abbreviations are used in the synthetic description:

AIBN, 2,2′-azobis(isobutyronitrile); Bn, benzyl; BOP-Cl,bis(2-oxo-3-oxazolidinyl)phosphinic chloride; Bu₂BOTf, dibutylborontriflate; CDI, N,N′-carbonyl-diimidazole; Cp, cyclopentadienyl; DBU,1,8-diazabicyclo-[5.4.0]undec-7-ene; DCC dicyclohexylcarbodiimide; DDQ,2,3-dichloro-5,6-dicyano-1,4-benzoquinone; DEAD,diethylazodicarboxylate; DIBAL-H diisobutylaluminum hydride; DMF,dimethylformamide; Gilbert reagent, dimethyl diazomethylphosphonate;LDA, lithium diisopropylamide; LiHMDS, lithium hexamethyldisilazamide;mesylate, methanesulfonic acid ester; Mitsunobu reagents, (DEAD, PPh₃,and nucleophile); NMO, N-methylmorpholine-N-oxide; Ph, phenyl; PhFl,9-phenyl-9-fluorenyl; Ph₂NTf, N-phenyltrifluoromethanesulfonimide; Swernoxidationreagents ((COCl)₂, DMSO, Et₃N); TBAF, tetrabutylammoniumfluoride; TBS, tert-butyldimethylsilyl; TCDI,thiono-N,N′-carbonyldiimidazole; Tf₂O, trifluoromethane-sulfonic acidanhydride; TMS, trimethylsilyl; triflate, trifluromethane sulfonateester; and TsCl, p-toluene-sulfonyl chloride.

The following reagents are also used:

The invention is based, in part, on the structure-function informationdisclosed herein which suggests the following preferred stereochemicalrelationships. Note that a preferred compound may have one, two, three,or more stereocenters having the indicated up-down (or β-α where β asdrawn herein is above the plane of the page) or (R)-(S) relationship. Insome embodiments, preferred configurations are 7(R), 9(S), 6(S), orcombinations thereof. In lactacystin analogs, R² and L^(O) preferablyhave a syn relationship to allow formation of a lactone or lactam-typeintermediate, which is believed to be related to the activity of thecompounds disclosed herein as proteasome inhibitors. However, it is notrequired that every stereocenter conform to the structures below.

A person of skill will recognize that the compounds described hereinpreserve certain stereochemical and electronic characteristics found ineither lactacystin or lactacystin β-lactone. For example, thehydroxyisobutyl group and the configuration of the hydroxyl group on C9are believed to be important for recognition of the target, as are theconfigurations of the C₆ hydroxyl and the C7 methyl of the γ-lactamring. However, the N-acetylcysteine moiety is not required for activity.Moieties such as R², R¹, and particularly A¹ (e.g., where A¹ is a sidechain of a naturally occurring α-amino acid such as Val, Leu, Lys, andPhe) can be modified to control selectivity for the proteasome, andselectivity for a particular peptidase activity of the proteasome. Incombination, these moieties simulate certain peptides or proteinsprocessed or degraded by the proteasome (i.e., are peptidomimetics).

The invention is also based, in part, on the finding that lactacystinand lactacystin β-lactone are highly selective for the X/MB1 subunit andα-chain of the proteasome and do not inhibit the activity of proteasessuch as trypsin, α-chymotrypsin, calpain I, calpain II, cathepsin, andpapain. Such selectivity is useful to formulate a pharmaceuticalcomposition with fewer side effects and to evaluate basic researchresults involving any subunit of the proteasome in conjunction with theselective inhibition of the X/MB1 subunit and α-chain.

Turning to the novel compounds described by the formulas of compoundscontained within the pharmaceutical compositions, several embodimentsare next considered.

Embodiments of the first aspect include compounds having one or more ofthe following limitations. In one limitation L¹ is linked by an oxygenor preferably sulfur atom to the carbon atom bonded to X². Such an L¹forms a thioester, thus providing a good leaving group. The precisenature of the thioester group is generally not critical. In initialexperiments, the magnitude of activity measured by K_(obs)/[I] for bothlarge and small thioesters was about the same, whether the thioesterterminus was a simple arylalkyl group or a more complex, branched moietywith intervening amide linkages and terminal carboxylic acid, esters, oraminocarbonyl groups. In one embodiment, Z¹ is NH or NR_(a); X¹ is O orS; Z² is CHR¹ or CHOR¹, R¹ being H, halogen, C₁₋₆ alkyl, or C₁₋₆haloalkyl; R² is OR_(f); and L⁰ is —(C═X²)—L¹, X² being O and L¹ beinglinked by a sulfur atom to the carbon atom bonded to X². In anotherembodiment, L¹ is selected from substituted or unsubstituted C₆₋₂₀arylalkylcarbonylthio, C₆₋₂₀ arylthio, C₂₋₈ alkylcarbonylthio, and C₁₋₁₂alkylthio. As defined above in the “Terms” section, arylthio includesarylthio, alkylarylthio, arylalkylthio, and alkylarylalkylthio groups.

In other limitations, A¹ is C₅₋₂₀ alkyl when L^(O) is carboxyl or (C₁₋₄alkyl)oxycarbonyl and A¹ is alkyl; only one of A¹ and L^(O) is selectedfrom carboxyl and (C₁₋₁₄ alkyl)oxycarbonyl; A¹ cannot be H; L¹ has atleast 3 carbon atoms when Z¹ and Z² are both NH, and A¹ is methoxy; whenR¹ and R² are taken together, Z² is CR¹; and when one of A¹ and L^(O) is(C₁₋₁₄ alkyl)oxycarbonyl or carboxyl, the other of A¹ and L^(O) has afragment formula weight of at least 20. Novel compounds of the first,fifth, and seventh aspects generally do not have A¹ being H.

Further embodiments of the first aspect, when Z¹ is NH and Z² is (R)CHR¹, are compounds wherein: L¹ has between 0 and 3 nonaromatic acycliccarbon atoms when there are 5 heteroatoms; L¹ has between 6 and 15nonaromatic acyclic carbon atoms when there is only one oxygen atom; andL¹ has 0, 1, 3, or 5 nonaromatic carbon atoms when there are threehalogen atoms.

Further embodiments of the first aspect include a compound wherein Z² isCHR¹ and Z¹ is NH or NR_(a); wherein L^(O) is C₂₋₁₆ oxiranyl; L¹ ishydroxy, C₁₋₁₂ alkoxy, C₁₋₁₂ alkyl-sulfonyloxy, C₆₋₂₀ arylsulfonyloxy,C₇₋₂₀ arylalkyl, C₆₋₂₀ aryloxy, C₆₋₂₀ arylalkylcarbonyloxy, C₂₋₈alkylcarbonyloxy, C₂₋₈ alkylcarbonylthio, C₁₋₁₂ alkylthio, C₆₋₂₀arylalkylcarbonylthio, or C₆₋₂₀ arylthio; or L² is H, C₁₋₂ haloalkyl, orC₁₋₆ alkyl. In addition, Z² is O, S, NH, or NR_(d). In anotherembodiment, Z² is O, S, NHJ, NR_(d), or CHR¹; and R_(h) is H, C₁₋₆alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, or C₂₋₆ alkynyl.

Further embodiments of the first aspect include compounds wherein Z¹ isO, S, or SO₂; wherein Z² is O, S, NH, or NR_(d); wherein X¹ is CH₂,CHR_(b), or C(R_(b))(R_(c)); wherein R² is C₁₋₆ alkyl, C₁₋₆ haloalkyl,C₂₋₆ alkenyl, or halogen; wherein R² is OR_(f), SR_(f), or NR_(f)R_(g);wherein Z² is in the beta (above plane of page) configuration; whereinCHX⁴ is in the alpha (below plane of paper) configuration; wherein R² isin the beta configuration; or combinations thereof.

The invention also provides a pharmaceutical composition wherein L^(O)is H or —(C═X²)—L¹, X² being O and L¹ being linked by an oxygen orsulfur atom to the carbon atom bonded to X²; where only one of A¹ andL^(O) is selected from carboxyl and (C₁₋₁₄ alkyl)oxycarbonyl; wherein L¹is hydroxy, C₁₋₁₂ alkoxy, C₁₋₁₂ alkylsulfonyloxy, C₆₋₂₀ arylsulfonyloxy,C₇₋₂₀ arylalkyl, C₆₋₂₀ aryloxy, C₆₋₂₀ arylalkylcarbonyloxy, C₂₋₈alkylcarbonyloxy, C₆₋₂₀ arylalkylcarbonylthio, C₆₋₂₀ arylthio, C₂₋₈alkylcarbonylthio, or C₁₋₁₂ alkylthio.

The invention also provides embodiments wherein Z¹ is NH or NR_(a); X¹is O or S; Z² is CHR¹ or CHOR¹, R¹ being H, halogen, C₁₋₆ alkyl, or C₁₋₆haloalkyl; R² is OR_(f); and L⁰ is —(C═X²)—L¹, X² being 0 and L¹ beinglinked by a sulfur atom to the carbon atom bonded to X². Individualcompounds include lactacystin, clasto-lactacystin trifluoroethyl ester,descarboxylactacystin, lactacystin amide, and phenylacetyl lactacystin.

Embodiments of the second aspect include compounds wherein CHX⁴ is inthe (S) configuration when X⁴ is hydroxyl and —(CH₂)_(n)—R³X³ isisopropyl; wherein the moiety —(CH₂)_(n)—R³X³ has between 5 and 20carbon atoms when X⁴ is hydroxyl, m is O, and Z¹ is NH; wherein Z¹ is NHor NR_(a); wherein Z¹ is O, S, or SO₂; wherein Z¹ is NR_(a); wherein X¹is CH₂, CH(R_(b)), or C(R_(b))(R_(c)); wherein Z² is in the beta (aboveplane of page) configuration; wherein CHX⁴ is in the alpha (below planeof paper) configuration; wherein R¹ is H, C₁₋₆ alkyl, C₁₋₆ haloalkyl,C₂₋₆ alkenyl, C₂₋₆ alkynyl, or halogen; wherein R¹ is a side chain of anaturally occurring α-amino acid, OR_(d), SR_(d), or NR_(d)R_(e); orcombinations of the above.

Another embodiment of the second aspect includes compounds wherein Z¹ isNH or NR_(a); X¹ is O or S; Z² is CHR¹ in the (R) or (S) configuration,wherein R¹ is H, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl,hydroxyl, halogen, a side chain of a naturally occurring amino acid,OR_(d), SR_(d), or NR_(d)R_(e) (each of R_(d) and R_(e), independently,being H, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, or C₂₋₅ alkynyl); Z³is O or NH; X² is O or S; and A¹ is the side chain of any naturallyoccurring α-amino acid, or is of the following formula,

—Y—(CH₂)_(n)—R³X³

wherein Y is C═O or CHX⁴ in the (R) or (S) configuration, or deleted, X⁴being halogen, methyl, halomethyl, or OR_(h), wherein R_(h) is selectedfrom H, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₁₀acyl, C₁₋₆ alkylsulfonyl, and C₆₋₁₀ arylsulfonyl; n is 0, 1, 2, or 3;and R³ is straight chain or branched C₁₋₈ alkylene, C₃₋₁₀ cycloalkylene,phenylene, C₆₋₁₄ arylalkylene, or deleted; and X³ is H, hydroxyl, thiol,carboxyl, amino, halogen, or C₆₋₁₀ aryl; or R³ and X³ taken together arethe side chain of any naturally occurring α-amino acid.

The invention also provides a compound wherein Z¹ is NH and Z³ is 0; ora compound wherein each of Z¹ and Z³ is NH; or a compound wherein atleast four (e.g., at least 5, or all) of the following limitationsapply: Z¹ is NH or NR_(a); X¹ is O or S; Z² is CHR¹ in the (R) or (S)configuration, wherein R¹ is H, C₁₋₆ alkyl, C₁₋₆ haloalkyl, hydroxyl, orOR_(d), R_(d) being C₁₋₆ alkyl; Z³ is O or NH; X² is O; Z³ is O or NH;and A¹ is the side chain of any naturally occurring α-amino acid, orCHX⁴, X⁴ being halogen, methyl, halomethyl, or OR_(h), R_(h) beingselected from H, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₁₀ acyl, C₁₋₆alkylsulfonyl, and C₆₋₁₀ arylsulfonyl; R³ is straight chain or branchedC₁₋₈ alkylene, C₃₋₁₀ cycloalkylene, phenylene, C₆₋₁₄ arylalkylene, ordeleted; m is 0, 1, or 2 and n is 0 or 1; and X³ is H.

The invention also provides a pharmaceutical composition wherein Z¹ isNH or NR_(a); X¹ is O or S; Z² is CHR¹ in the (R) or (S) configuration,wherein R¹ is H, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl,hydroxyl, halogen, a side chain of a naturally occuring α-amino acid,OR_(d), SR_(d), or NR_(d)R_(e) (each of R_(d) and R_(e), independently,being H, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, or C₂₋₅ alkynyl); Z³is O or NH; X² is O or S; and A¹ is the side chain of any naturallyoccurring α-amino acid, or is of the following formula,

—Y—(CH₂)_(n)—R³X³

wherein Y is C═O or CHX⁴ in the (R) or (S) configuration, or deleted, X⁴being halogen, methyl, halomethyl, or OR_(h), wherein R_(h) is selectedfrom H, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₁₀acyl, C₁₋₆ alkylsulfonyl, and C₆₋₁₀ arylsulfonyl; n is 0, 1, 2, or 3;and R³ is straight chain or branched C₁₋₈ alkylene, C₃₋₁₀ cycloalkylene,phenylene, C₆₋₁₄ arylalkylene, or deleted; and X³ is H, hydroxyl, thiol,carboxyl, amino, halogen, or C₆₋₁₀ aryl; or R³ and X³ taken together arethe side chain of any naturally occurring α-amino acid.

Another embodiment includes a compound wherein X² is O, and A¹ is theside chain of any naturally occurring α-amino acid, or X⁴ is halogen,methyl, halomethyl, or OR_(h), R_(h) being selected from H, C₁₋₆ alkyl,C₁₋₆ haloalkyl, C₁₋₁₀ acyl, C₁₋₆ alkylsulfonyl, and C₆₋₁₀ arylsulfonyl;R³ is straight chain or branched C₁₋₈ alkylene, C₃₋₁₀ cycloalkylene,phenylene, C₆₋₁₄ arylalkylene, or deleted; m is 0, 1, or 2 and n is 0 or1; and X³ is H. Individual lactone analogs include clasto-lactacystinβ-lactone.

Embodiments of the third aspect include compounds wherein: when X¹ is 2singly bonded H and R¹ has only one oxygen, then the fragment formulaweight of R¹ is at least 30 atomic mass units; when X¹ is 2 singlybonded H and R¹ is 2H, and A¹ is alkyl, then A¹ has at least 3 carbonatoms; A¹, R¹ and X¹ taken together have at least one carbon atom,halogen, or heteratom; when X¹ is two singly bonded H, and R¹ is H, X²is O, and R_(h) is alkyl, then R_(h) is C₄₋₆ alkyl; and when n is 2,R_(h) is C₁₋₆ haloalkyl.

Further embodiments of the third aspect include compounds wherein: X¹ isCH₂, two singly bonded H, CH(R_(b)) or C(R_(b))(R_(c)); Z² is O or S; Z²is NH or NR_(j); R¹ is C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, C₂₋₆alkynyl, or halogen; R¹ is a side chain of a naturally occuring α-aminoacid, OR_(d), SR_(d), or NR_(d)R_(e); A¹ cannot be H; R¹ is in the betaconfiguration; X⁴ is in the alpha configuration; or combinationsthereof.

Embodiments of the fourth aspect include compounds wherein: Z² is O orS; Z² is NH or NR_(j); X¹ is O or S; R¹ is C₁₋₆ alkyl, C₁₋₆ haloalkyl,C₂₋₆ alkenyl, C₂₋₆ alkynyl, or halogen; R¹ is a side chain of anaturally occuring α-amino acid, OR_(d), SR_(d), or NR_(d)R_(e);; R² isH, C₁₋₃ alkyl, C₁₋₃ haloalkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl, C₆₋₁₂ aryl,C₃, heteroaryl, or halogen; R² is H, C₃₋₆ alkyl, C₃₋₆ haloalkyl, C₄₋₆alkenyl, C₄₋₆ alkynyl, C₆₋₁₂ aryl, or C₃₋₈ heteroaryl; A¹ cannot be H;R¹ is in the beta configuration; X⁴ is in the alpha configuration; orcombinations of the above.

Further embodiments of the fifth aspect are compounds wherein: Z¹ is Oor S; Z² is NH or NR_(j); R¹ is H, C₁₋₃ alkyl, C₁₋₃ haloalkyl, C₂₋₃alkenyl, C₂₋₃ alkynyl, or a side chain of a naturally occuring α-aminoacid, OR_(d), SR_(d), or NR_(d)R_(e); R¹ is H, C₄₋₆ alkyl, C₄₋₆haloalkyl, C₄₋₆ alkenyl, C₄₋₆ alkynyl, halogen, or a side chain of anaturally occuring α-amino acid, OR_(d), SR_(d), or NR_(d)R_(e); R² isH, C₁₋₃ alkyl, C₁₋₃ halo-alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl, C₆₋₁₂ aryl,C₃₋₈ heteroaryl, or halogen; R² is H, C₄-6 alkyl, C₄₋₆ haloalkyl, C₄₋₆alkenyl, C₄₋₆ alkynyl, C₆₋₁₂ aryl, or C₃₋₈ heteroaryl; n R_(a) is C₁₋₃alkyl; A¹ cannot be H; X⁴ is in the alpha configuration; or combinationsthereof.

Embodiments of the sixth aspect are compounds wherein: X¹ is O or S; X¹is CH₂, two singly bonded H, CH(R_(b)), or C(R_(b)) (R_(c)); Z¹ is O orS; Z¹ is NH or NR_(a); R¹ is C₁₋₃ alkyl, C₁₋₃ haloalkyl, C₂₋₃ alkenyl,C₂₋₃ alkynyl, halogen, or a side chain of any naturally occuring α-aminoacid; R¹ is OR_(d), SR_(d), or NR_(d)R_(e); R¹ is C₄₋₆ alkyl, C₄₋₆haloalkyl, C₄₋₆ alkenyl, C₄₋₆ alkynyl, a side chain of any naturallyoccuring α-amino acid, OR_(d), SR_(d), or NR_(d)R_(e); R¹ and R² takentogether are a bivalent moiety; R¹ is in the beta configuration; R² isin the beta configuration; R² is C₁₋₃ alkyl, C₁₋₃ haloalkyl, C₆₋₁₂ aryl,C₃₋₈ cycloalkyl, or halogen; R² is C₄₋₆ alkyl, C₄₋₆ haloalkyl, C₆₋₁₂aryl, C₃₋₈ cycloalkyl, C₃₋₈ heteroaryl, or C₃₋₈ heterocyclic radical; A¹has a higher fragment formula weight than A²; A¹ has a lower fragmentformula weight than A²; A¹ is in the (R) configuration; A¹ is in the (S)configuration; only one of A¹ and A² is H; A¹ is a side chain of anynaturally occuring α-amino acid; or combinations thereof.

Embodiments of the seventh aspect are compounds wherein: X¹ is O or S;X¹ is CH₂, two singly bonded H, CH(R_(b)) or C(R_(b))(R_(c)); Z² is O orS; Z² is NH or NR_(j); R¹ is C₁₋₃ alkyl, C₁₋₃ haloalkyl, C₂₋₃ alkenyl,C₂₋₃ alkynyl, hydroxyl, halogen, or a side chain of any naturallyoccuring α-amino acid; R¹ is OR_(d), SR_(d), or NR_(d)R_(e); R¹ is C₄₋₆alkyl, C₄₋₆ haloalkyl, C₄₋₆ alkenyl, C₄₋₆ alkynyl, a side chain of anynaturally occuring α-amino acid; R¹ and R² taken together are a bivalentmoiety; R² is C₁₋₃ alkyl, C₁₋₃ haloalkyl, C₂₋₃ alkenyl, azido, C₂₋₃alkynyl, hydroxyl, or halogen; R² is OR_(f), SR_(f), NR_(f)R_(f)R_(g),—ONR_(f)R_(g), —NR_(g)(OR_(f)), or —NR_(g)(RS_(f)); R² is C₄₋₆ alkyl,C₄₋₆ haloalkyl, C₄₋₆ alkenyl, azido, C₄₋₆ alkynyl, OR_(f), SR_(f),NR_(f)R_(f)R_(g), —ONR_(f)R_(g), —NR_(g)(OR_(f)), or —NR_(g)(RS_(f)); R¹and R² taken together are a bivalent moiety; R¹ is in the alphaconfiguration; R¹ is in the beta configuration; R² is in the alphaconfiguration; R² is in the beta configuration; A¹ is in the alphaconfiguration; A² is in the beta configuration; or combinations thereof.

One embodiment of the eighth aspect is a compound where A¹, A², R¹², andX¹ taken together have at least one carbon atom and one heteratom.Further embodiments of the eighth aspect are compounds wherein: Z¹ isNH, or NR_(a); X¹ is O or S; X¹ is CH₂, or two singly bonded H; R¹² isH, C₁₋₃ alkyl, C₁₋₃ haloalkyl, C₂₋₃ alkenyl, or C₂₋₃ alkynyl; R¹² isC₄₋₆ alkyl, C₄₋₆ haloalkyl, C₄₋₆ alkenyl, or C₄₋₆ alkynyl; where A¹, A²,R¹², and X¹ taken together have at least one carbon atom and oneheteratom; where the fragment formula weight of A¹ is greater than thefragment formula weight of A² (e.g., by at least 30 or 60); where thefragment formula weight of A² is greater than the fragment formulaweight of A¹ (e.g., by at least 15 or 50); or combinations thereof.

Embodiments of the ninth aspect are compounds wherein A¹ is a side chainof any naturally occurring α-amino acid; wherein A¹ has a fragmentformula weight of at least 50 (e.g., 70, 100, or 120).

Examples of individual compounds described herein are provided in TablesA, B, and C.

Synthesis

The syntheses disclosed herein are organized by structure groups. Aperson of skill will recognize that compounds of claim 1 are related tothe compounds in the compositions of claim 23, and the compounds ofclaim 5 are related to the compounds in the compositions of claim 30 andso on. The synthesis of the compounds in claims 1-5 rely primarily onthe results reported by Uno, et al. in their enantiospecific synthesisof (+)-lactacystin from (R)-glutamate [Uno, et al., J. Am. Chem. Soc.(1994) 116:2139]. Note that in all schemes relating to claims 1-5 and24-32, when a straight line is used to connect A¹ (or anythingcorresponding to A¹) to the rest of the molecule, a dashed line shouldbe assumed. The straight line is used simply for clarity. All A¹ areattached to the rest of the molecule on the “alpha,” i.e., the bottom,face.) Compound A-1 (Scheme 1) serves as the starting material for allsynthetic routes proposed in claim 2, for example. Thus, conversion ofA-1 to A-2, in analogy to the above-mentioned work allows forintroduction of R¹ via standard alkylation chemistry. In the cases whereR¹ is hydroxyl, halide, —SR_(d), or —NR_(d)R_(e), wherein R_(d) andR_(e) each, independently, is selected from H, C₁₋₆ alkyl, C₁₋₆haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, the electrophile in step b is,respectively, a N-arylsulfonyl-3-phenyloxaziridine [Davis, F. A. et al.,J. Org. Chem. (1984), 49:3241], a N-halosuccinimide [Stotter, P. A. etal., J. Org. Chem. (1973) 38:2576], R_(d)S—SR_(d) or elemental sulfur[Zoretic, P. A. et al., J. Org. Chem. (1976) 41:3587] [Gassman, P. G. etal., J. Org. Chem. (1977) 42:3236], a N-arylsulfonylazide (followed byreduction of the azide to the primary amine and elaboration toNR_(d)R_(e)) [Evans, D. A. et al., J. Am. Chem. (1987) 109:6881]. If R¹is labile or reactive, or both, a protecting group would be introducedat this point and removed at the end of the synthesis. The preparationof compounds of type A-2 is then completed with the two-step (steps band c) process reported by Uno [Uno, et al., (1994)].

Introduction of A¹ is the next task. Uno [Uno, et al., (1994)] showedthat under Lewis-acid mediated aldol addition conditions, A¹ isintroduced with very high facial selectivity with respect to thebicyclic aminoacid derivative A-2. This carbon-carbon bond constructionallows for the A¹ groups detailed in claim 2. Accordingly, conversion ofA-2 to A-3, as described by Uno,

followed by treatment of A-3 with SnCl₄ and an aldehyde (R_(f)CHO, whereR_(f) corresponds to H or to the rest of A¹), in diethylether yieldsaldol products A-4 (or A-5 if R_(f) is H), following suitable protectionof the hydroxyl group with a group designated here as P_(o). [Greene andWuts, et al., Protective Groups in Organic Synthesis, 2nd ed., (1991)].

Introduction of R² by a stereoselective, conjugate nucleophilic additionto the unsaturated carbonyl system of A-4 or A-5, followed by quenchingof the resulting enolate with acidic water gives access to compounds A-6and A-7, respectively. This process is analogous to the “three-componentcoupling” strategy for prostaglandin synthesis of [Suzuki, et al.(1988)]. In this case, the three components are A-4 (or A-5), R², and aproton. The diastereomers shown for A-6 and A-7 are predicted to be themajor ones based on several facts. The Uno synthesis [Uno, H., et al.,(1994)] used an Os0₄-catalyzed dihydroxylation of a compound similar toA-4 (R¹=methyl, R_(f)=iso-propyl) to introduce a hydroxyl groupcorresponding to R². This reaction proceeded with complete facialselectivity. Thus, in general, groups with appropriate reactivity(nucleophiles or electrophiles) similarly approach preferentially the“beta” face (from the top) of A-4 or A-5.

Quenching of the enolate with acidic water proceeds stereoselectively.In Suzuki's three-component coupling strategy, [Suzuki, et al., (1988)]the nucleophile and electrophile are introduced trans to each other. Inour case, R² and the proton are introduced trans to each other. In theevent that compounds in which R¹ and R² are trans to each other aredesired, as depicted in compounds A-12 and A-13, a similar strategy hasbeen devised.

The basic strategy has one exception to that detailed above: The orderof introduction of R¹ and proton at the carbon alpha to the carbonyl isreversed. Thus, conversion of A-1 to A-9 is carried out using the sameprocedures as those for the preparation of A-3, omitting the alkylationstep (step a). Elaboration of A-9 to A-10 and A-11 utilizes the sameconditions as those which afford A-4 and A-5. Addition of R² stilloccurs from the top face, but the quenching step with an electrophilecorresponding to R¹ now gives a trans relationship of R¹ and R², asshown in compounds A-12 and A-13.

Another advantage of these approaches to A-6, A-7, A-12, and A-13 is asfollows. Should the quenching step introduce the electrophile (proton orR¹) cis to R² (opposite that predicted above), then one still hasstereospecific routes to the desired compounds, except that A-4 and A-5give A-12 and A-13, respectively, and A-10 and A-11 give A-6 and A-7.

Compounds in which A¹ is H are prepared by a different strategy (Scheme2). For example, it has been shown that when A¹ is H, nucleophiles[Hanessian, et al., Synlett (1991) 10:222] and electrophiles[Griffart-Brunet, et al., Tet. Lett. (1991) 35:119] approach from thebottom face, cis to the hydrogen at Al. Thus, the key to our strategyabove is that the stereoselectivity of addition is reversed when A¹ isnot H. The rapid preparation described below of compounds in which A¹ isH takes advantage of this reversal of stereoselectivity.

Thus, bromination of A-2 by standard methods [Caine, et al., J. Org.Chem. (1985) 50:2195] gives A-14, which is utilized in conjugateaddition-elimination procedures to give compounds of type A-15. Since A¹is H, catalytic hydrogenation of the olefin should install R¹ and R² inthe desired cis configuration shown in compound A-16. Cleavage of theN,O-aminal may occur under such conditions, giving compound A-17. Suchan occurrence is not disadvantageous since such a deprotection is thenext step in the synthesis anyway. Compounds A-18 and A-19 are accessedby base-catalyzed epimerization of A-16 and A-17, respectively.

The above strategies allow for the preparation of compounds containingany and all R¹ and R² described in claims 2 and 25, with one exception:compounds where an additional ring is fused to the lactam ring(specifically, compounds in which R¹ is taken together with R², giving abivalent moiety which forms a C₃₋₈ cycloalkyl, C₃₋₈ heteroaryl, C₃₋₈heterocyclic radical, or C₆₋₁₂ aryl). Their preparation will bediscussed below (Schemes 3 and 4). Note that while only carbocyclicsystems are depicted in Schemes 3 and 4, both the alicyclic andheterocyclic (aromatic and non-aromatic in both cases) classes ofcompounds can be prepared in this manner by choice of the appropriatereagents. Further, all substitution patterns on these rings that arenormally accessible by such processes are accessible by the methodsproposed below. The simplest carbocyclic variants are shown for clarityand to illustrate the general strategy to these compounds.

The synthesis of these compounds relies on the diverse chemistry ofcycloaddition [Carruthers, et al., Cycloaddition Reactions in organicSynthesis (1990) and J. March, Advanced organic Chemistry, (1992) pp.826-877] to alpha-beta unsaturated olefins. Thus, as shown in Scheme 3,[1+2] processes (e.g. diazoinsertion, cyclopropanation, epoxidation, andaziridination) with compound A-20 (see Scheme 1 for preparation ofcompounds of this type, e.g. A-10 and A-11) gives access to compounds oftype A-21. Compounds of type A-22 are prepared with a variety of [2+2]cycloaddition processes (e.g. photocycloadditions and ketene additions).1,3-Dipolar cycloaddition chemistry ([3+2] cycloaddtion reagents, suchas nitrile oxides and azomethine ylides) prepares a wide varietycompounds of the type A-23. The preparation of compounds of type A-24 isperhaps the most flexible and far-reaching of the cycloadditionchemistry

because it relies on the Diels-Alder reaction, one of the most utilizedand studied reactions in all of organic chemistry. The variety ofcompounds of type A-24 that are prepared by this method is far to greatto detail; [Carruthers, (1990)] however, notably, bridged compounds(type A-25) are prepared by this method. [5+2] cycloadditions to giveA-26 are less common, but some intriguing examples have been reported.[Wender, et al., J. Org. Chem. (1991) 56:6267 and Wender, et al., Tet.Lett. (1992) 32:6115].

While all these procedures introduce R¹ and R² cis to each other,base-catalyzed epimerization in the cases of A-24 and A-26 gives accessto the trans-fused versions of these compounds, A-28 and A-29,respectively. (A-21, A-22, A-23, and A-25 can not be epimerized in thisfashion, as these trans-fused rings suffer from much more ring strainthan do the cis-fused systems.)

Cycloheptyl (A-26) and cyclooctyl (A-27) compounds are prepared with aslightly different process, one that is akin to that shown in Scheme 1.Fundamentally, one adds an nucleophile (abbreviated as “Nu”) to A-20which also has a leaving group (abbreviated as “LG”) attached to theother end of the incoming nucleophile, separated by the necessary numberof atoms (5 in the case of A-26, 6 in the case of A-27). Thus, followingthe addition of the nucleophile, the resulting enolate displaces theleaving group, forming the ring. Alternatively, such a process is donestepwise in order to minimize undesired intramolecular side reactions ofthe nucleophile and electrophile. These and other methods for makingcycloheptyl and cyclooctyl compounds from alpha-beta unsaturatedcarbonyl compounds have been reviewed in the chemical literature.[Petasis, N. A., et al., Tetrahedron (1992) 48:5757].

Aryl and heteroaryl compounds (e.g. compounds A-30 and A-31, Scheme 4)are prepared by [4+2] cycloaddition chemistry, followed by oxidation ofthe resultant cycloalkene or heterocycloalkene withdichlorodicyanoquinone (DDQ). [Pizey, N., Synth. Reagents (1977)3:193-225]. In the case of compounds of type A-31, other types of dienesthat are utilized are the hydroxyorthoxylylenes, formed by the ringopening reaction of hydroxybenzocyclobutenes. [Arnold, B. J., et al., J.Chem. Soc., (1974) 409-4153.

Thus, having prepared all the variations of R¹ and R², we turn now tothe elaboration of these compounds to include all of the variations ofA¹ (Scheme 5).

Based on the results of [Uno, et al., (1994)] and as shown in Scheme 1,Lewis-acid catalyzed aldol additions of A-3 to R_(f)CHO, followed byfurther elaboration, provide compounds of the type A-33 (Scheme 5) withthe stereocenters in the configurations shown, including bothconfigurations of R¹. (A straight bond (i.e. neither bold or dashed) isshown here to represent both diastereomers at this position.) Here wewill focus on the stereochemistry of the secondary hydroxyl that isincluded in A¹, and related derivatives. While modification ofconditions in the aldol addition (Scheme 1) reaction can provide theother diastereomer at this position, another method of inverting thiscenter is to first oxidize to the ketone under standard conditions (e.g.Swern, Dess-Martin periodinane, etc.) and then reduce with theappropriate reducing agent (e.g. NaBH₄, etc.) whose identity isdetermined by screening a number of reductants. This two-step procedureprovides compounds of type A-35. Analogously, reductive amination ofketone A-34 with an amine or hydroxylamine (represented in Scheme 5 asR_(n)NH₂) and sodium cyanoborohydride (NaCNBH₃) provides compounds oftype

A-36. The sense of stereochemical induction in this step can be alteredby using a different reducing agent. Alternatively, by first preparingthe methanesulfonate ester (“mesylate”) of A-33 or A-35, and thendisplacing the mesylate with a nucleophile (e.g. RNH₂, RS—, RO—, halide,hydroxylamine, Grignard reagents, etc.) one obtains a wide variety of A¹derivatives with complete control of stereochemistry at the carboncorresponding to the secondary hydroxyl in A-33. Reduction of themesylate (with lithium triethylborohydride (LiEt₃BH)) or of a halide(with, e.g., zinc in hydrochloric acid) provides compounds of the typeA-38.

Another method to produce the various A¹ derivatives is depicted inScheme 6. Compounds of the type A-39, which are prepared according toScheme 1, are converted to those of type A-40 where X_(a) is a halide ortrifluoromethanesulfonate ester (“triflate”), for example, usingstandard functional group manipulation. Displacement of these leavinggroups with nucleophiles gives another method for the preparation ofcompounds of type A-38. Subjecting iodide A-40 (X_(a) is I) tometal-halogen exchange conditions (e.g. activated Mg metal ortert-butyllithium) provides nucleophiles to which a variety ofnucleophiles can be added, providing yet another route to compounds oftype A-38.

Another strategy for the preparation of a subset of compounds of type 33is worth noting (Scheme 7). This strategy allows for the preparation ofcompounds of type A-33 for all R² discussed above and for all R¹ otherthan hydroxyl, halide, —SR_(d),—NR_(d)R_(e) Compounds of the type A-4,A-5, A-10, and A-11 as shown in Scheme 1 (represented as compound A-41in Scheme 7), in which R¹ is attached to the lactam ring with acarbon-carbon bond, can be dihydroxylated to give compounds of typeA-42, in which the hydroxyl groups have been added to the top faceexclusively. [Uno, et al., (1994)] (The reasons for thisstereoselectivity have been discussed above.) Following the procedure of[Uno, et al., 1994)] the tertiary hydroxyl can be removed selectively,yielding a mixture of R¹ diastereomers A-43, which can be separated by avariety of chromatographic methods. Mitsunobu inversion of the secondaryhydroxyl, followed by displacement of a suitably derived leaving groupby any nucleophile corresponding to R² (e.g. hydroxide, alkoxide,sulfide, Grignard reagents, amine, halide, etc.) gives compounds of thetype A-44, which are a subset of compounds of the type A-33, and cantherefore be elaborated into their corresponding A¹ derivatives asdescribed above.

Thus, having provided viable routes to all variations of R¹, R², and nowA¹ in claims 2 and 25, we now turn our attention to the installation ofX¹, L¹, and X² (Scheme 8).

The compound of general structure A-45 can be converted to A-46 in atwo-step process involving first catalytic hydrogenolysis or mild acidichydrolysis [Corey, E. J., et al., J. Am. Chem. Soc. (1992a), 114:10677]and then protection of the primary hydroxyl group as itstert-butyldimethylsilyl (TBS) ether under standard conditions. [Corey,E. J. and Venkateswarlu, A., J. Am. Chem. Soc. (1972), 94:6190]N-Alkylation of the amide provides access to compounds A-47. [Challis,N., The Chemistry of Amides, (1970) 734-754]. (In the cases where R_(b)is desired to be H, the amide can be blocked with a suitable protectinggroup that will be removed at the end of the synthesis. [Greene, T. W.,et al., Protective Groups in Organic Synthesis (1991)].

Enamine A-48 can be prepared from A-47 under Tebbe olefinationconditions. [Pine, S. H., et al., J. Org. Chem. (1985) 50:1212].Thioamide A-49 can be prepared bytreatment of A-47 withbis(tricyclohexyltin)sulfide and

boron trichloride. [Steliou, K., et al., J. Am. Chem. Soc., (1982)104:3104]. Substituted enamines A-50 can be prepared by addition of theappropriate Grignard reagent or alkyllithium to A-47 or A-49, followedby acidic hydrolysis, and separation of the E and Z olefin isomers.[Aguerro, A., et al., J. Chem. Soc., Chem. Commun. (1986) 531 andSchrock, R. R., J. Am. Chem. Soc., (1976) 98:5399, and Hansen, C., J.Org. Chem. (1973) 38:3074]. Pyrrolidines of type 51 can be prepared byreduction of A-47 with lithium aluminum hydride [March, J., AdvancedOrganic Chemistry (1992) 826-877 and Gaylord, J., Reduction With ComplexMetal Hydrides (1956)] 544-636] or alternatively by reduction of A-49with Raney nickel. [Belen'kii, W., Chemistry of Organosulfur Compounds91990) 193-228].

Taken together, these procedures constitute a synthesis of compounds ofthe general structure A-52, which are converted to A-53 following, forexample, the procedure of Uno, et al. [Uno, H., et al., (1994)] (Scheme9). Compounds A-53 are converted to analogues A-54a via oxidation to theacid and coupling with L¹ (all of which can be prepared by standardmethods) under the conditions utilized by Corey and Reichard [Corey, E.J., et al., (1992a]. Sulfurization of these compounds with Lawesson'sreagent [Cava, M. P., et al., Tetrahedron (1985) 41:5061] gives thionoanalogues A-55a. Compounds A-54b are prepared via addition of anucleophile (e.g. CF₃) [Francese, C., et al., J. Chem. Soc., Chem.Commun (1987) 642], corresponding to L² to the aldehydes A-54b followedby oxidation of the alcohol with Dess-Martin periodinane. [Linderman, R.J., et al., Tet. Lett. (1987) 28:42593. Sulfurization of these compoundswith Lawesson's reagent [Cava, M. P., et al., (1985)] gives thionoanalogues A-55b. Epoxides of the type A-55c are prepared from A-53 orA-54b by the method of Johnson

[Johnson, C. R., Acc. Chem. Res. (1973) 6:341], thuscompleting thesynthesis of all the analogues detailed in this section.

The preparation of the compounds covered by claims 4 and 31 reliesprimarily on the methods of Seebach and of Corey. Thus, as shown inScheme 10, epoxides of the type A-58 are prepared according to theprocedure of Corey, et al., [Corey, E. J., et al., (1992b)] from 61 viacompound 62. R² is thus installed by choosing the appropriate aldehydefor the aldol reaction in step a. Corey [Corey, E. J., et al., (1992b)]showed that epoxides A-58 are opened stereospecifically to give A-59.Thus, Z¹ is installed by treating A-58 with a nucleophile correspondingto Z¹. (P_(z) refers to a protecting group for Z¹.) Installation of Z²is accomplished by conversion of the hydroxyl A-59 to a leaving group,for example, a tosylate, and displacement of the leaving group with anucleophile corresponding to Z². (P_(z), refers to a protecting groupfor Z²).

The results of Seebach [Seebach, D., et al., Helv. Chim. Acta. (1987)70:1194] are used in the next part of the synthesis. Conversion of A-60to A-61 allows for enolization and alkylation with an electrophilecorresponding to A¹, yielding A-62. Seebach showed that such alkylationsgive predominantly the diastereomer shown.

After reduction of the ester and protection of the resulting primaryalcohol as the TBS ether, removal of the tert-butylmethylene protectinggroup, enables conversion to compounds of either the type A-63 (bytreatment with, for example, formaldehyde and an acid catalyst) or thetype A-64 (by treatment with, for example, CDI) is effected.

Compound is a key intermediate in the completion of thesynthesis of thecompounds in claims 4 and 31, (Scheme 11). Compounds of the type A-65are prepared from A-64 under Tebbe olefination conditions. [Pine, S. H.,et al., (1985)]. Compounds of the type A-66 are prepared by treatment ofA-64 with bis(tricyclohexyltin)sulfide and boron trichloride. [Steliou,K., et al., (1982)]. Compounds of the type A-67 are prepared by additionof the appropriate Grigard reagent or alkyllithium to A-64 or A-66,followed by acidic hydrolysis, and separation of the E and Z olefinisomers. [Aguerro, A., et al., (1986) and Schrock, R. R., (1976), andHansen, C., et al., (1973)]. Compounds of the type A-63, prepared inScheme 10, are also prepared by reduction of A-64 with lithium aluminumhydride [March, J., (1992), and Gaylord, J. (1956)] or alternatively byreduction of A-66 with Raney nickel. [Belen'kii, W., (1990)].

Taken together, compounds of the type A-63, A-64, A-65, A-66, and A-67are of the general class A-68, which are converted the general class oflactacystin analogues A-69 by fluoride deprotection of the TBS ether,oxidation of the resulting primary alcohol to the carboxylic acid, viathe aldehyde analogues, in, for example, the two-step process shown, andcoupling with Li using the method of Corey and Reichard, [Corey, E. J.,et al., (1992a)] and removal of the protecting groups on Z¹ and Z² (ifnecessary). Lactacystin analogues A-71 are prepared by treating A-69with Lawesson's reagent [Cava, M. P., et al., (1985)]. Analogues A-70are also prepared from A-68 by fluoride deprotection of the TBS ether,oxidation of the resulting primary alcohol to the aldehyde, addition ofnucleophile (e.g., CF₃) [Francese, C. et al. 1987, supra] Dess-Martinperiodinane oxidation, sulfurization with Lawesson's reagent (ifdesired) [Cava, M. P., 91985)], and deprotection of Z² and Z², ifnecessary. Epoxides of the type A-72 are also prepared from A-68 byfluoride deprotection of the TBS ether, oxidation of the

resulting primary alcohol to the aldehyde, addition of a nucleophilecorresponding to L², Dess-Martin periodinane oxidation, addition ofdimethyloxosulfonium methylide, and deprotection of Z¹ and Z² (ifnecessary). Thus, the above strategy prepares all the compounds inclaims 4 and 31.

The preparation of the compounds covered by claim 1 relies primarily onthe methods of Evans and of Corey. Thus, as shown in Scheme 12, chiraloxazolidinones of the type A-73 are enolized and alkylated withbenzyloxy-bromomethane, thereby installing R¹. [Evans, D. A., et al. J.Am. Chem. Soc., (1982) 104:1737]. (Only one configuration of R¹ isobtained in the reaction. The other configuration of R¹ is obtained byusing the opposite enantiomer of the chiral oxazolidinone.) Conversionof A-74 to aldehydes of the type A-75, and chiral, boron-mediated (usingA-76) aldol condensation with tert-butylbromoacetate gives [Corey, E.J., et al., (1992b)] a secondary alcohol that is protected as thetert-butyldimethylsilyl (TBS) ether to give compounds of the type A-77.Displacement of the bromide with a nucleophile [Corey, E. J., et al.(1992b)] corresponding to Z¹ yields A-78, which can be converted tocompounds of the type A-79. As discussed above regarding claim 4, suchcompounds can be enolized and alkylated stereospecifically withelectrophiles corresponding to A¹, giving A-80. Standard functionalgroup manipulation yields tosylates of the type A-81. Displacement ofthe tosylate with a nucleophile corresponding to R², [Hanessian, S., etal., J. Org. Chem. (1989) 54:5831] gives compounds of the type A-82, theprimary alcohol of which is deprotected via, for example, catalytichydrogenolysis, and cyclized to compounds A-83 after oxidation of theprimary alcohol to the carboxylic acid.

As shown in Scheme 13, compounds of the type A-84, in which R¹ is ineither the (R) or (S) configuration, are converted into a variety of X¹variants. Compounds of the type A-85 are prepared from A-84 under Tebbeolefination conditions. [Pine, et al., (1985)] Compounds of the typeA-86 are prepared by treatment of A-84 with bis(tricyclohexyltin)sulfideand boron trichloride. [Stelious, K., et al., (1982)]. Compounds of thetype A-87 are prepared by addition of the appropriate Grignard reagentor alkyllithium to A-84 or A-86 followed by acidic hydrolysis, andseparation of the E and Z olefin isomers. [Aguerro, A., et al., (1986),Schrock, R. R., (1976) and Hansen C., et al., (1973)]. Compounds of thetype A-88 are prepared by reduction of A-84 with lithium aluminumhydride [March, J. (1992), and Gaylord, J., (1956)] or alternatively byreduction of A-86 with Raney nickel. [Belen'kii, W., (1990)]. When Z¹ isS in A-88, oxidation to the cyclic sulfone variants is effected with,for example, KHSO₅. [Trost, B. M., et al., Tet. Lett. (1981) 22:1287].

Taken together, compounds of the type A-84, A-85, A-86, A-87, A-88, andA-89 are of the general class A-90, which are converted analogues A-91,following for example, the procedure of Uno, et al. [Uno, H., et al.,(1994)] (Scheme 13b). Compounds A-91 are converted to analogues A-92 viaoxidation to the acid and coupling with L¹ (all of which can be preparedby standard methods) under the conditions utilized by Corey andReichard. [Corey, E. J., et al., (1992a)]. Sulfurization of thesecompounds with Lawesson's reagent gives thiono analogues A-93. CompoundsA-94 are prepared via addition of a nucleophile (e.g. CF₃) [Francese,C., et al., (1987)] corresponding to L² to the aldehydes A-91 followedby oxidation of the alcohol with Dess-Martin periodinane. Sulfurizationof these compounds

with Lawesson's reagent gives thiono analogues A-95. Epoxides of thetype A-96 are prepared from A-91 or A-94 by the method of Johnson, thuscompleting the synthesis of all the analogues detailed in this section.Note: As above in all Schemes relating to claim 6, when a straight lineis used to connect A² (or anything corresponding to A²) to the rest ofthe molecule, a dashed line should be assumed. The straight line is usedsimply for clarity. All A² are attached to the rest of the molecule onthe “alpha” face (i.e. the bottom face).

The proposed syntheses of all of the compounds listed in claim 17 relyon a key intermediate from Scheme 9. As shown in Scheme 14, compounds oftype B-1 are analogous to a subset of compounds A-53, whose variouspreparations were described in Schemes 1, 2, and 5-8, and described inthe accompanying text.

In order to prepare analog B-2, the method of Corey, et al. is utilized.[Corey, E. J., et al., Tet. Lett. (1993) 34:6977]. Sulfurization of B-2with, for example, Lawesson's reagent [Cava, M. P., et al., (1985)]provides analogs B-3.

The preparation of compounds of type B-1 is summarized in Scheme 15.Thus the same starting material (A-1) as that used in Scheme 1 iselaborated to all of the compounds B-2 by the same methods as those usedin Schemes 1, 2, 5, 6, and 8. There are then two possible generalstrategies for the conversion of these intermediates to B-2. The firstis analogous to that detailed in Schemes 1, 2, 5, 6, 8, and 9, in whichZ⁵ (suitably activated or protected) serves as the nucleophile in therelevant reactions.

The other strategy, based on that shown in Scheme 7 is also depicted inScheme 15. Thus, dihydroxylation of B-4 gives B-5, which is then bedeoxygenated as before, giving B-6. Separation of the diastereomers,Mitsunobu

inversion of the secondary hydroxyl, and displacement of a leaving groupderived from the Mitsunobu product provides compounds of the type B-7,which are then elaborated to B-1 as described in Schemes 8 and 9.

The proposed syntheses of all of the compounds listed in claim 17 relyon intermediate A-81 from Scheme 12. As shown in Scheme 16, compounds oftype B-li are analogous to a subset of compounds A-81, whose variouspreparations are illustrated in Scheme 12, and described in theaccompanying text. Conversion of compounds of the type B-11 to B-12 isperformed in analogy to Scheme 12. Compounds of the type B-13 areprepared from B-12 following the relevant steps in Schemes 13a and 13b.

In order to prepare analogs B-14, the method of Corey, et al. isutilized. [Corey, E. J., et al., (1993)] Sulfurization of B-14 with, forexample, Lawesson's reagent (Cava, M. P., et al., (1985)] providesanalogs B-15.

The preparation of the compounds covered by claim 7 relies on a keyintermediate from Scheme 7. As shown in Scheme 17, compound C-1 isanalogous to A-41, whose preparation is illustrated in Scheme 7 anddescribed in the accompanying text. Following the work of Fuchs,[Hutchinson, D. K., et al., J. Am. Chem. Soc. (1987)] C-1 is treatedsequentially with bis(benzyloxymethyl)lithium cuprate and acidic water,giving compounds of the type C-2. Both configurations of R⁷ can beaccessed. See Schemes 1 and 2 for details. As in Scheme 8, mild acidichydrolysis, protection of the primary hydroxyl, and alkylation orprotection of the nitrogen gives compounds C-2.

Deprotection of the benzyl group by, for example, catalytichydrogenolysis followed by displacement of the derived tosylate with anucleophile corresponding to Z⁷, accesses compounds C-3, in which Z⁷ isprotected, if

necessary. Elaboration to carboxylic acids C-4 follows the same set ofreactions in Schemes 8 and 9, allowing the preparation of all X⁹variants. Deprotection of Z⁷, if necessary, followed by cyclizationwith, for example, DCC, yields bicyclic structures C-5 which if desiredare sulfurized with Lawesson's reagent [Cava, M. P., et al., (1985)] togive C-6.

The preparation of the compounds covered by the right-hand structure inclaim 7 relies on a key intermediate from Scheme 9. As shown in Scheme18, compound C-11 is analogous to a subset, defined by Z⁷, of the classof compounds A-52, whose preparation is illustrated in Schemes 1-8, anddescribed in the accompanying text. Deprotection of the primary hydroxylwith, for example, TBAF in THF and oxidation via, for example, underSwern oxidation conditions to the aldehydes C-12.Homologation of thealdehyde with, for example, the phosphorous ylide derived fromtriphenylmethoxymethylphosphonium chloride [Jamison, T. F., et al., J.Am. Chem. Soc., (1994) 116:5505] yields enolethers of the type C-13,which are converted to carboxylic acids C-14 with acidic hydrolysis andoxidation of the resulting aldehyde with, for example, buffered NaClO₂oxidation. [Corey, E. J., et al., (1992a)].

Deprotection of Z⁷, if necessary, followed by cyclization with, forexample, DCC, [Klausner, Y. S. et al., Synthesis (1972) 453] yieldsbicyclic structures C-15 which if desired are sulfurized with Lawesson'sreagent [Cava, M. P., (1985)] to give C-16.

The preparation of the compounds covered by claims 8 and 39 reliesprimarily on the boron-enolate mediated asymmetric aldol procedures ofEvans [Evans, D. A., et al., J. Am. Chem. Soc., (1981) 103:2127] and onthe results of studies on intramolecular radical

cyclizations. [Curran, D. P., Comprehensive organic Synthesis, (1991)4:779-831].

Esters C-21 (Scheme 19), prepared by standard methods, are deprotonatedand then alkylated with benzyloxybromo-methane and reduced with DIBAL-Hto give aldehydes C-22 as a racemic mixture. Addition of C-22 to anboron enolate derived from chiral oxazolidinone imides C-23 [Evans, D.A., et al., (1981)] yields C-24 as a mixture of A³ diastereomers. Theconfigurational identity of the other two new stereocenters isestablished in this reaction and is not affected by the #3 configurationof A³.

Conversion to aldehydes C-25 in a six-step procedure (for example,tosylation of the secondary hydroxyl, displacement with a nucleophilecorresponding to Z⁷, catalytic hydrogenolysis of the benzyl group,oxidation of the alcohol to the carboxylic acid and esterification)allows for preparation of the substrate for the intramolecular radicalcycloaddition. Thus, treatment of the aldehyde with the Gilbert reagent[Gilbert, J. C., et al., J. Org. Chem. (1982) 47:1837] and replacementof the enolizable hydrogen with iodine gives an acetylenes C-26, which,after deprotection of Z⁷, if necessary, and cyclization with, forexample, BOP-Cl, affords C-27.

Exposure of C-27 to atom-transfer, intramolecular radical cyclizationconditions [Curran, D. P., et al., Tet. Lett. (1987) 28:2477 and Curran,D. P., et al., J. Org. Chem. (1989) 54:3140] gives compounds C-28. Thisreaction deserves further comment. The stereochemistry of A¹ and theiodine atom are inconsequential because the radical generated from theiodine can interconvert under the reaction conditions. Further, only oneof the radical diastereomers will be able to cyclize—that giving thecis-4,5 ring system depicted, because the trans-4,5 suffers from muchgreater ring strain.

This cyclization is favored for two other reasons. First, it is a5-endo-dig type cyclization and therefore favored according to Baldwin'srules for cyclization. [Baldwin, J. E., J. Chem. Soc., Chem. Commun.(1976) 734]. Second, the atom transfer conditions used are ideal becausethe resulting vinylic radical is very reactive and is rapidly quenchedby the iodine radical. [Curran, D. P., et al., J. Am. Chem. Soc., (1986)108:2489].

Elaboration of C-28 to C-29 is accomplished using, for example, standardStille [Stille, J. K., Angew. Chem., Int. Ed. Engl. (1986) 25:504] orHeck [Heck, R. F., Comprehensive Organic Synthesis, (1991) 4:833-863]type coupling conditions using a suitable metallic derivative (M is,e.g., tributylstannyl) of R⁹. Sulfurization with Lawesson's reagent[Cava, M. P., et al., (1985)] gives compounds C-30, thus completing thepreparation of all the compounds claimed in claims 8 and 39.

The preparation of the compounds covered by claims 9 and 40 reliesprimarily on intermediate B-2, whose preparation is described in Schemes13a, 13b and 14 and discussed in the accompanying text. As shown inScheme 20, C-31 is deprotonated with a suitable base, and the resultingenolate is treated with, for example, the triflate source PhN(Tf)₂[McMurry, J. E., et al., Tet. Lett. (1983) 24:979]. The vinyl triflatesC-32 are treated with, for example, a catalytic amount of (Ph₃P)₄Pd anda suitable metallic derivative [Stille, J. K. (1986) and Heck, R. F.(1991)] (M is, e.g., tributylstannyl) of R⁹, giving analogues C-33,which can be treated with Lawesson's reagent [Cava, M. P., et al.,(1985)] gives compounds C-34, thus completing the preparation of all thecompounds in this section.

The preparation of the compounds covered by claims 10 and 41 reliesprimarily on intermediate A-38, whose

preparation is illustrated in Schemes 1, 5, and 6, anddiscussed in theaccompanying text. Compounds of the type C-41 (Scheme 21) can thereforebe prepared with either the (R) or (S) configuration of Z⁷ by followingthe procedures illustrated in Schemes 1-6, and discussed in theaccompanying text.

Mild acidic hydrolysis of C-41, [Corey, E. J., et al., (1992a)] followedby protection of the primary hydroxyl [Corey, E. J., et al., (1972)]allows for installation of A⁴ via standard alkylation procedures,[Challis, N. (1970)] giving C-42. Elaboration of these intermediates toC-43 is performed as described for the analogous compounds in Scheme 8.Deprotection of the primary hydroxyl allows for oxidation to thecarboxylic acids C-44, which are cyclized with, for example, BOP-Cl,giving analogues C-45, which can be treated with Lawesson's reagent[Cava, M. P., et al., (1985)] gives compounds C-46, thus completing thepreparation of all the compounds in claims 10 and 41.

The preparation of the compounds covered by claims 11 and 42 follows thestrategy illustrated in Schemes 1, 3, 4, 5, 8, and 9, and described inthe accompanying text. Thus, as shown in Scheme 22, C-51, beinganalogous to A-2 is elaborated to C-52 following the same methods usedin Scheme 1 using an aldehyde containing A⁵ and a protected form of Z⁷in either the (R) or (S) configuration. Protection of the secondaryhydroxyl gives compounds C-52, [Corey, E. J., et al., (1972)] which canbe treated with a nucleophile corresponding to R⁹ and subsequentquenching with acidic water, giving C-53. The reasons governing theindicated stereoselectivity of the nucleophilic addition and aqueousquenching have been discussed in the text accompanying Scheme 1.

Deprotection and subsequent conversion of the secondary hydroxyl to thetosylate allows for

introduction of A³ via displacement of the tosylate with a nucleophilecorresponding to A³ to give compounds of the type C-54. [Hanessian, S.,(1980)]. Elaboration of these compounds to C-55 is performed with themethods illustrated in Scheme 8 and described in the accompanying text.Deprotection of the primary hydroxyl allows for oxidation to thecarboxylic acids C-56, which are cyclized with, for example, DCC,[Klausner, Y. S., et al., (1972)] following deprotection of Z⁷, ifnecessary, to give analogues C-57. Sulfurization with Lawesson's reagent[Cava, M. P., (1985)] gives compounds C-58, thus completing thepreparation of all the compounds in claims 12 and 42.

The preparation of the compounds covered by claims 13 and 43 reliesprimarily on the results of Lubell, et al., J. Org. Chem., 1990 55:3511.Thus, as shown in Scheme 23, L-serine (C-61) is converted to ketonesC-62 by known procedures. (PhFl is an abbreviation of9-phenyl-9-fluorenyl.) Standard Wadsworth-Emmons olefination of C-62yields either C-63 or C-64, or a mixture thereof. The composition of theproduct mixture is of no consequence, since both olefin isomers yieldthe same diastereomer in the next reaction.

Thus, a cuprate reagent derived from A¹ approaches from the back face ofboth C-63 or C-64 since the very bulky PhFl group blocks the other facecompletely, giving C-65 as the major diastereomer. C-65 is converted toC-66 in a five-step procedure (reduction of the methyl ester to thealdehyde and protection as the dimethyl acetal, deprotection of theoxazolidinone, and oxidation of the primary alcohol to the carboxylicacid.)

As depicted in Scheme 24, DCC-mediated coupling, for example, of C-66with an amine, yields amides C-67. Taken together, C-66 and C-67comprise the general class of compounds C-88. The PhFl group is removedby

catalytic hydrogenolysis, and the free amine alkylated with anelectrophile corresponding to R¹² (reductive amination with an aldehydeor ketone and NaCNBH₃ is another option) to give compounds C-69. Acidichydrolysis of the dimethyl acetal yields intermediate aldehyde C-70,which cyclizes to analogues C-73.

Tebbe olefination of C-71 provides compounds C-73. Treatment of C-71with Lawesson's reagent provides compounds C-72, which can bedesulfurized with Raney Nickel to give compounds C-74, thus completingthe synthesis of all the compounds in claims 13 and 43.

The preparation of the compounds covered by claims 14 and 44 reliesprimarily on the results of Dener, et al., [J. Org. Chem., 1993,58:1159], and on those of Evans et al.,[J. Am. Chem. Soc., 1981,103:2127]. Thus, as shown in Scheme 25, D-aspartic acid (C-81) isprotected and alkylated with an electrophile corresponding to A⁶ to givecompounds C-82, with the major diastereomer to be that shown. Dener etal.,[J. Org. Chem., 1993, 58:1159]. (PhFl is an abbreviation of9-phenyl-9-fluorenyl.) Site-specific DIBAL-H reduction (owing to theextreme size of the PhFl group) affords aldehyde C-83.

A diastereoselective boron-mediated aldol addition is the next step.Following the procedure of Evans, et al., J. Am. Chem. Soc., (1981)103:2127, one obtains C-85, after TBS protection of the secondaryhydroxyl. The oxazolidinone is removed with lithium hydroperoxide, thePhFl group removed with catalytic hydrogenolysis, and lactam formationaccomplished under, for example, DCC conditions, to give C-86. Removalof the TBS group with TBAF and lactone formation (saponification of themethyl ester with LiOH, followed by DCC coupling may be necessary)yields C-87, which is elaborated to C-88 by alkylating the amidenitrogen with a nucleophile corresponding to R_(d). Epimerization withDBU provides access to compounds C-89. Treatment of C-89 with Lawesson'sreagent, Cava et al., Tetrahedron, 1985, 41: 5061, yields compoundsC-90, thus completing the preparation of all compounds in this section.Examples 13-21 below are individual syntheses.

Use

The disclosed compounds are used to treat conditions mediated directlyby the proteolytic function of the proteasome such as muscle wasting, ormediated indirectly via proteins which are processed by the proteasomesuch as NF-κB. The proteasome participates in the rapid elimination andpost-translational processing of proteins (e.g., enzymes) involved incellular regulation (e.g., cell cycle, gene transcription, and metabolicpathways), intercellular communication, and the immune response (e.g.,antigen presentation). Specific examples discussed below includeβ-amyloid protein and regulatory proteins such as cyclins andtranscription factor NF-κB. Treating as used herein includes reversing,reducing, or arresting the symptoms, clinical signs, and underlyingpathology of a condition in manner to improve or stabilize the subject'scondition.

Alzheimer's disease is characterized by extracellular deposits ofβ-amyloid protein (β-AP) in senile plaques and cerebral vessels. β-AP isa peptide fragment of 39 to 42 amino acids derived from an amyloidprotein precursor (APP). At least three isoforms of APP are known (695,751, and 770 amino acids). Alternative splicing of mRNA generates theisoforms; normal processing affects a portion of the β-AP sequence,thereby preventing the generation of β-AP. It is believed that abnormalprotein processing by the proteasome contributes to the abundance ofβ-AP in the Alzheimer brain. The APP-processing enzyme in rats containsabout ten different subunits (22 kDa-32 kDa). The 25 kDa subunit has anN-terminal sequence of X-Gln-Asn-Pro-Met-X-Thr-Gly-Thr-Ser, which isidentical to the β-subunit of human macropain. Kojima, S. et al., Fed.Eur. Biochem. Soc., (1992) 304:57-60. The APP-processing enzyme cleavesat the Gln¹⁵—Lys¹⁶ bond; in the presence of calcium ion, the enzyme alsocleaves at the Met⁻¹-Asp¹ bond, and the Asp¹-Ala² bonds to release theextracellular domain of β-AP.

One embodiment, therefore, is a method of treating Alzheimer's disease,including administering to a subject an effective amount of a compound(e.g., pharmaceutical composition) having a formula disclosed herein.Such treatment includes reducing the rate of β-AP processing, reducingthe rate of β-AP plaque formation, and reducing the rate of β-APgeneration, and reducing the clinical signs of Alzheimer's disease.

Other embodiments of the invention relate to cachexia and muscle-wastingdiseases. The proteasome degrades many proteins in maturingreticulocytes and growing fibroblasts. In cells deprived of insulin orserum, the rate of proteolysis nearly doubles. Inhibiting the proteasomereduces proteolysis, thereby reducing both muscle protein loss and thenitrogenous load on kidneys or liver. Proteasome inhibitors are usefulfor treating conditions such as cancer, chronic infectious diseases,fever, muscle disuse (atrophy) and denervation, nerve injury, fasting,renal failure associated with acidosis, and hepatic failure. See, e.g.,Goldberg, U.S. Pat. No. 5,340,736 (1994). Embodiments of the inventiontherefore encompass methods for: reducing the rate of muscle proteindegradation in a cell, reducing the rate of intracellular proteindegradation, reducing the rate of degradation of p53 protein in a cell,and inhibiting the growth of p53—related cancers). Each of these methodsincludes the step of contacting a cell (in vivo or in vitro, e.g., amuscle in a subject) with an effective amount of a compound (e.g.,pharmaceutical composition) of a formula disclosed herein.

Another protein processed by the proteasome is NF-κB, a member of theRel protein family. The Rel family of transcriptional activator proteinscan be divided into two groups. The first group requires proteolyticprocessing, and includes p50 (NF-κB1, 105 kDa) and p52 (NF-κ2, 100 kDa).The second group does not require proteolytic processing, and includesp65 (RelA, Rel (c-Rel), and RelB). Both homo- and heterodimers can beformed by Rel family members; NF-κB, for example, is a p50-p65heterodimer. After phosphorylation and ubiquitination of IκB and p105,the two proteins are degraded and processed, respectively, to produceactive NF-κB which translocates from the cytoplasm to the nucleus.Ubiquitinated p105 is also processed by purified proteasomes. Palombellaet al., Cell (1994) 78:773-785. Active NF-κB forms a stereospecificenhancer complex with other transcriptional activators and, e.g., HMGI(Y), inducing selective expression of a particular gene.

NF-κB regulates genes involved in the immune and inflammatory response,and mitotic events. For example, NF-κB is required for the expression ofthe immunoglobulin light chain κ gene, the IL-2 receptor α-chain gene,the class I major histocompatibility complex gene, and a number ofcytokine genes encoding, for example, IL-2, IL-6, granulocytecolony-stimulating factor, and IFN-β. Palombella et al., (1994). Someembodiments of the invention include methods of affecting the level ofexpression of IL-2, MHC-I, IL-6, IFN-β or any of the otherpreviously-mentioned proteins, each method including administering to asubject an effective amount of a compound of a formula disclosed herein.

NF-κB also participates in the expression of the cell adhesion genesthat encode E-selectin, P-selectin, ICAm, and VCAM-1, Collins, T., Lab.Invest. (1993) 68:499-508. One embodiment of the invention is a methodfor inhibiting cell adhesion (e.g., cell adhesion mediated byE-selectin, P-selectin, ICAm, or VCAM-1), including contacting a cellwith (or administering to a subject) an effective amount of a compound(e.g., pharmaceutical composition) having a formula disclosed herein.

NF-κB also binds specifically to the HIV-enhancer/promoter. Whencompared to the Nef of mac239, the HIV regulatory protein Nef of pbj14differs by two amino acids in the region which controls protein kinasebinding. It is believed that the protein kinase signals thephosphorylation of I-κB, triggering IκB degradation through theubiquitin-proteasome pathway. After degradation, NF-κB is released intothe nucleus, thus enhancing the transcription of HIV. Cohen, J.,Science, (1995) 267:960. Two embodiments of the invention are a methodfor inhibiting or reducing HIV infection in a subject, and a method fordecreasing the level of viral gene expression, each method includingadministering to the subject an effective amount of a compound of aformula disclosed herein.

Complexes including p50 are rapid mediators of acute inflammatory andimmune responses. Thanos, D. and Maniatis, T., Cell (1995) 80:529-532.Intracellular proteolysis generates small peptides for presentation toT-lymphocytes to induce MHC class I-mediated immune responses. Theimmune system screens for autologous cells that are virally infected orhave undergone oncogenic transformation. Two embodiments of theinvention are a method for inhibiting antigen presentation in a cell,including exposing the cell to a compound of a formula described herein,and a method for suppressing the immune system of a subject (e.g.,inhibiting transplant rejection), including administering to the subjectan effective amount of a compound of a formula described herein.

In addition, the invention provides a method for treating inflammation,wherein the method includes administering to a subject an effectiveanti-inflammatory amount of a pharmaceutical composition containing acompound of a formula described herein. Inflammation can be a primary orsecondary responses associated with (a) injury such as a cut,laceration, puncture wound, (b) infection (including infected surgicalincisions) by one or more viruses, bacteria, mycobacteria,microorganisms, parasites, and fungi, (c) allergies, (d) a diseasestate, (e) surgery (e.g., transplantation), or (f) a combinationthereof.

Allergies are primary inflammatory responses to antigens or allergens.Sources of allergens include plants (e.g., grass or tree pollen),animals (e.g., dander, venom, urine, execreta from dogs, cats, insects,and snakes), and fungi. In addition to allergens such as rye grass,ragweed, and Japanese cedar pollen, certain foods or food components(e.g., eggs, milk, shellfish, strawberries, chocolate), vaccines, anddrugs (e.g., penicillin) can induce allergic reactions in certainindividuals.

Disease states include rheumatoid arthritis, scleroderma, rheumaticfever, inflammatory bowel disease (e.g., Crohn's disease and ulcerativecolitis), diabetes mellitus, myasthenia gravis, multiple sclerosis,Guillain-Barre syndrome, conjuctiva of the eye, systemic lupuserythematosus, encephalitis, Adult Respiratory Distress Syndrome,psoriasis, emphysema, Alzheimer's disease, and muscular dystrophy.

The invention provides a method of treating inflammation induced byorgan or tissue transplantation. This method includes administering to apatient who has undergone or is about to undergo transplantation acomposition containing a compound having a formula disclosed herein.Transplantations include bone marrow, solid organ (e.g., kidney, lungs,heart, pancreas, liver, and skin), or tissues.

Certain proteasome inhibitors block both degradation and processing ofubquitinated NF-κB in vitro and in vivo. Proteasome inhibitors alsoblock IκB-α degradation and NF-κB activation, Palombella, et al.; andTraenckner, et al., EMBO J. (1994) 13:5433-5441. One embodiment of theinvention is a method for inhibiting IκB-α degradation, includingcontacting the cell with a compound of a formula described herein. Afurther embodiment is a method for reducing the cellular content ofNF-κB in a cell, muscle, organ, or subject, including contacting thecell, muscle, organ, or subject with a compound of a formula describedherein.

Other eukaryotic transcription factors that require proteolyticprocessing include the general transcription factor TFIIA, herpessimplex virus VP16 accessory protein (host cell factor), virus-inducibleIFN regulatory factor 2 protein, and the membrane-bound sterolregulatory element-binding protein 1.

Other embodiments of the invention are methods for affectingcyclin-dependent eukaryotic cell cycles, including exposing a cell (invitro or in vivo) to a compound of a formula disclosed herein. Cyclinsare proteins involved in cell cycle control. The proteasome participatesin the degradation of cyclins. Examples of cyclins include mitoticcyclins, G1 cyclins, (cyclin B). Degradation of cyclins enables a cellto exit one cell cycle stage (e.g., mitosis) and enter another (e.g.,division). It is believed all cyclins are associated with p34^(cdc2)protein kinase or related kinases. The proteolysis targeting signal islocalized to amino acids 42-RAALGNISEN-50 (destruction box). There isevidence that cyclin is converted to a form vulnerable to a ubiquitinligase or that a cyclin-specific ligase is activated during mitosis.Ciechanover, A., Cell, (1994) 79:13-21. Inhibition of the proteasomeinhibits cyclin degradation, and therefore inhibits cell proliferation(e.g., cyclin-related cancers). Kumatori et al., Proc. Natl. Acad. Sci.USA (1990) 87:7071-7075. One embodiment of the invention is a method fortreating a proliferative disease in a subject (e.g., cancer, psoriasis,or restenosis), including administering to the subject an effectiveamount of a compound of a formula disclosed herein. Chronic or acuteinflammation can result from transplantation rejection, arthritis,rheumatoid arthritis, infection, dermatosis, inflammatory bowel disease,asthma, osteoporosis, and autoimmune diseases. Rejection or inflammationcan occur in transplanted tissues or organs of any type, includingheart, lung, kidney, liver, skin grafts, and tissue grafts. Theinvention also encompasses a method for treating cyclin-relatedinflammation in a subject, including adminstering to a subject aneffective amount of a compound of a formula described herein.

Additional embodiments are methods for affecting theproteasome-dependent regulation of oncoproteins and methods of treatingor inhibiting cancer growth, each method including exposing a cell (invivo, e.g., in a subject or in vitro) to a compound of a formuladisclosed herein. HPV-16 and HPV-18-derived E6 proteins stimulate ATP-and ubiquitin-dependent conjugation and degradation of p53 in crudereticulocyte lysates. The recessive oncogene p53 has been shown toaccumulate at the nonpermissive temperature in a cell line with amutated thermolabile E1. Elevated levels of p53 may lead to apoptosis.Examples of proto-oncoproteins degraded by the ubiquitin system includec-Mos, c-Fos, and c-Jun. One embodiment is a method for treatingp53-related apoptosis, including administering to a subject an effectiveamount of a compound of a formula disclosed herein.

Treatment of cancer prevents, alleviates, or ameliorates one or moreprimary or secondary phenomena associated with the initiation,progression, and metastasis of tumors, especially malignant tumors,e.g., a growth of tissue wherein cell multiplication is uncontrolled.Malignant tumors show a greater degree of anaplasia than do benigntumors. The invention provides a method of treating cancer includingadministering to a subject an effective anti-cancer amount of apharmaceutical composition described herein, wherein the cancer isselected from carcinoma, lymphoma, sarcoma, and myeloma.

Examples of carcinomas include adenocarcinoma, acinic celladenocarcinoma, adrenal cortical carcinomas, alveoli cell carcinoma,anaplastic carcinoma, basaloid carcinoma, basal cell carcinoma,bronchiolar carcinoma, bronchogenic carcinoma, renaladinol carcinoma,embryonal carcinoma, anometroid carcinoma, fibrolamolar liver cellcarcinoma, follicular carcinomas, giant cell carcinomas, hepatocellularcarcinoma, intraepidermal carcinoma, intraepithelial carcinoma,leptomanigio carcinoma, medullary carcinoma, melanotic carcinoma,menigual carcinoma, mesometonephric carcinoma, oat cell carcinoma,squamal cell carcinoma, sweat gland carcinoma, transitional cellcarcinoma, and tubular cell carcinoma. Examples of sarcoma includeamelioblastic sarcoma, angiolithic sarcoma, botryoid sarcoma,endometrial stroma sarcoma, ewing sarcoma, fascicular sarcoma, giantcell sarcoma, granulocytic sarcoma, immunoblastic sarcoma, juxaccordialosteogenic sarcoma, coppices sarcoma, leukocytic sarcoma (also known asleukemia), lymphatic sarcoma (also known as lympho sarcoma), medullarysarcoma, myeloid sarcoma (also known as granulocytic sarcoma),austiogenci sarcoma, periosteal sarcoma, reticulum cell sarcoma (alsoknown as histiocytic lymphoma), round cell sarcoma, spindle cellsarcoma, synovial sarcoma, and telangiectatic audiogenic sarcoma.Examples of lymphomas include Hodgkin's disease and lymphocyticlymphomas, such as Burkitt's, nodular poorly-differentiated lymphocytic(NPDL), nodular mixed lymphocytic (NML), NH (nodular histiocytic), anddiffuse lymphomas. Additional carcinomas include neural blastoma,glioblastoma, astrocytoma, melanoma, leiomyo sarcoma, multiple myeloma,and Hemangioma.

A tripeptide aldehyde protease inhibitor (benzyloxycarbonyl(Z)—Leu-Leu-leucinal induces neurite outgrowth in PC12 cells at anoptimal concentration of 30 nM, Tsubuki et al., Biochem. and Biophys.Res. Comm. (1993) 196:1195-1201. Peptide aldehydes have been shown toinhibit the chymotryptic activity of the proteasome. Vinitsky, et al.,1992, Tsubuki et al., 1993. One embodiment of the invention is a methodof promoting neurite outgrowth, including administering to the subject acompound of a formula disclosed herein.

Finally, the disclosed compounds are also useful as diagnostic agents(e.g., in diagnostic kits or for use in clinical laboratories) forscreening for proteins (e.g., enzymes, transcription factors) processedby the proteasome. The disclosed compounds are also useful as researchreagents for specifically binding the X/MB1 subunit or α-chain andinhibiting the proteolytic activities associated with it. For example,the activity of (and specific inhibitors of) other subunits of theproteasome can be determined.

Most cellular proteins are subject to proteolytic processing duringmaturation or activation. Lactacystin can be used to determine whether acellular, developmental, or physiological process or output is regulatedby the proteolytic activity of the proteasome. One such method includesobtaining an organism, an intact cell preparation, or a cell extract;exposing the organism, cell preparation, or cell extract to a compoundof a formula disclosed herein; exposing the compound-exposed organism,cell preparation, or cell extract to a signal, and monitoring theprocess or output. The high selectivity of the compounds disclosedherein permits rapid and accurate elimination or implication of theproteasome in a given cellular, developmental, or physiological process.

The compounds and compositions of the invention are useful in severalmethods including a method of treating inflammation, comprisingadministering to a subject an effective anti-inflammatory amount of apharmaceutical composition described herein (e.g., a lactacystinthioester, a lactone, or a lactam); wherein the inflammation isassociated with injury or infection; wherein the inflammation isassociated with an allergy or asthma; wherein the inflammation isassociated with a disease state selected from rheumatoid arthritis,scleroderma, rheumatic fever, inflammatory bowel disease, diabetesmellitus, myasthenia gravis, multiple sclerosis, Guillan-Barre syndrome,conjunctiva of the eye, systemic lupus erythematosus, encephalitis,Adult Respiratory Distress Syndrome, emphysema, Alzheimer's disease, andmuscular dystrophy; wherein the inflammation is associated withtransplantation of bone marrow or a solid organ selected from kidney,lung, heart, pancreas, liver, and skin, and the composition isadministered before, during, or after transplantation; wherein thepharmaceutical composition is administered orally; or combinationsthereof.

The invention also provides a method of treating cancer, comprisingadministering to a subject an effective anti-cancer amount of apharmaceutical composition described herein; a method for treatingpsoriasis, comprising administering to a subject an effective amount ofa pharmaceutical composition described herein; and a method for treatingrestenosis, comprising administering to a subject an effective amount ofa pharmaceutical composition described herein. Preferred compounds andcompositions include various lactacystin thioesters, and lactacystinP-lactone analogs, including the p-lactone itself.

Formulation and Administration

The methods of the invention contemplate treatment of animal subjects,such as mammals (e.g., higher primates, and especially humans). Theinvention encompasses pharmaceutical compositions which include novelcompounds described herein, and pharmaceutical compositions whichinclude compounds described and first recognized herein as proteasomeinhibitors, such as lactacystin.

Pharmaceutically acceptable salts may be formed, for example, with 1, 2,3, or more equivalents of hydrogen chloride, hydrogen bromide,trifluoroacetic acid, and others known to those in the art of drugformulation. Compounds of the invention can be formulated intopharmaceutical compositions by admixture with pharmaceuticallyacceptable non-toxic excipients and carriers. A pharmaceuticalcomposition of the invention may contain more than one compound of theinvention, and/or may also contain other therapeutic compounds notencompassed by the invention, such as anti-inflammatory, anti-cancer, orother agents. A subject may have more than one type of inflammation, ormore than one type of cancer, a combination of allergies, or a mixtureof the above conditions for which the disclosed compounds are useful. Acompound of the invention may be administered in unit dosage form, andmay be prepared by any of the methods well known in the pharmaceuticalart, for example, as described in Remington's Pharmaceutical Sciences(Mack Pub. Co., Easton, Pa., 1980). The invention also encompasses apackaged drug, containing a pharmaceutical composition formulated intoindividual dosages and printed instructions for self-administration.

Compounds disclosed herein as proteasome inhibitors may be prepared foruse in parenteral administration in the form of solutions or liquidsuspensions; for oral administration (preferable), particularly in theform of tablets or capsules; or intranasally, particularly in the formof powders, gels, oily solutions, nasal drops, aerosols, or mists.Formulations for parenteral administration may contain as commonexcipients sterile water or sterile saline, polyalkylene glycols such aspolyethylene glycol, oils of vegetable origin, hydrogenatednaphthalenes, and the like. Controlled release of a compound of theinvention may be obtained, in part, by use of biocompatible,biodegradable polymers of lactide, and copolymers of lactide/glycolideor polyoxyethylene/polyoxypropylene. Additional parental deliverysystems include ethylene-vinyl acetate copolymer particles, osmoticpumps, implantable infusion systems, and liposomes. Formulations forinhalation administration contain lactose, polyoxyethylene-9-laurylether, glycocholate, or deoxycholate. Formulations for buccaladministration may include glycocholate; formulations for vaginaladministration may include citric acid.

The concentration of a disclosed compound in a pharmaceuticallyacceptable mixture will vary depending on several factors, including thedosage of the compound to be administered, the pharmacokineticcharacteristics of the compound(s) employed, and the route ofadministration. In general, the compounds of this invention may beprovided in an aqueous physiological buffer solution containing about0.1-10% w/v of compound for parenteral administration. Typical doseranges are from about 0.1 to about 50 mg/kg of body weight per day,given in 1-4 divided doses. Each divided dose may contain the same ordifferent compounds of the invention. The dosage will be an effectiveamount depending on several factors including the overall health of apatient, and the formulation and route of administration of the selectedcompound(s).

The effective amount of the active compound used to practice the presentinvention for treatment of conditions directly or indirectly mediated bythe proteasome varies depending upon the manner of administration, theage and the body weight of the subject and the condition of the subjectto be treated, and ultimately will be decided by the attending physicianor veterinarian. Such amount of the active compound as determined by theattending physician or veterinarian is referred to herein as “effectiveamount”.

Without further elaboration, it is believed that one skilled in the artcan, based on the description herein, utilize the present invention toits fullest extent. The following specific examples are, therefore, tobe construed as merely illustrative, and not limitative of the remainderof the disclosure in any way whatsoever. All publications cited hereinare hereby incorported by reference.

EXAMPLES Example 1

Synthesis of [³H] Lactacystin

Lactacystin was prepared from the β-lactone, which was tritiated by anoxidation-reduction sequence. Unlabeled β-lactone was oxidized at C9 tothe ketone with the Dess-Martin periodinane in dichloromethane and thenreduced with [³H]NaBH₄ (NEN, 13.5 Ci/mmol) in 1,2-dimethoxyethane/1% H₂Oto afford tritiated β-lactone along with its C9-epimer (2:3 ratio). Theisomeric β-lactones were separated by HPLC (Rainin Microsorb SiO₂column, 4.6×100 mm, 10% i-Pr-OH in hexane) and reacted separately withN-acetylcysteine (0.5 M) and Et₃N (1.5 M) in CH₃CN to afford thelactacystin (9S) and its C9-epimer (9R), which were purified from theirrespective reaction mixtures by reverse-phase HPLC (TSK ODS-80T_(m)column, 4.6×250 mm, 10% CH₃CN/0.1% CF₃CO₂H in H₂O).

Example 2

Fluorography of Crude Cell and Tissue Extracts

Crude Neuro 2A extracts (˜11 μg total protein/lane) and crude bovinebrain extracts (˜54 μg total protein/lane) were treated as follows: (1)10 μM [³H]lactacystin; (2) 10 μM [³H]lactacystin and 1 mM coldlactacystin; (3) 10 μM [³H]lactacystin; (4) 10 μM [³H]lactacystin and 1mM cold lactacystin; (5) 10 μM [³H]lactacystin and 1 mM cold β-lactone;(6) 10 μM [³H]lactacystin and 1 mM cold phenylacetyl lactacystin; (7) 10μM [³H]lactacystin and 1 mM cold lactacystin amide; (8) 10 μM[³H]lactacystin and 1 mM cold dihydroxy acid; (9) 10 μM [³H]lactacystinand 1 mM cold 6-deoxylactacystin; (10) 10 μM [³H]lactacystin and 1 mMcold (6R,7S)-lactacystin (6-epi, 7-epi); (11) 10 μM [³H]lactacystin and1 mM cold des(hydroxyisobutyl)-lactacystin. Crude extracts from Neuro2Aneuroblastoma cells or bovine brain were incubated in the presence of 10μM [³H]lactacystin in the presence or absence of 1 mM cold competitor(added simultaneously) for 24 h at room temperature, followed bySDS-polyacrylamide gel electrophoresis (0.75-mm-thick 12% polyacrylamidegel). The gel was stained in 0.1% Coomassie brilliant blue R-250, 12%acetic acid, 50% MeOH, and then destained in 12% acetic acid, 50% MeOH,followed by impregnation withEN³HANCE (NEN) and precipitation of thefluor with water. The gel was dried on Whatman filter paper in a geldryer at 65° C. for 30 min and then exposed to Kodak SB film at −78° C.

Example 3

Separation of Purified Protein Complex (20S proteasome)

Bovine brain was frozen in liquid N₂. A total of two kg of brain was dryhomogenized in a Waring blenderfor one minute. All operations wereperformed at 4° C., except as noted. The following buffer (6 literstotal) was then added (pH 7.7): 18.25 mM K₂HPO₄, 6.75 mM KH₂PO₄, 0.27 Msucrose, 2 mM EDTA, 2 mM EGTA, 25 mM NaF, 5 mM tetrasodiumpyrophosphate, 5 μg/ml each of leupeptin and pepstatin A, and 5 mMβ-mercaptoethanol. The tissue was wet homogenized in the Waring blenderfor two minutes. The homogenate was centrifuged at 5,000 g for 15 minand then at 12,000 g for 30 min. Ammonium sulfate was added to thesupernatant to 50% saturation, and the sample spun at 10,000 g for 20min. The 50-60% saturation ammonium sulfate fraction, containing thelactacystin-binding activity as determined by SDS-PAGE and fluorographyof samples incubated with radioactive compound, was then dialyzedovernight against 20 mM MES—NaOH, pH 5.6, 5 mM β-mercaptoethanol,followed by SP-sepharose chromatography (Pharmacia SP-sepharose, fastflow; 120-ml bed volume column) with 20 mM MES—NaOH, pH 5.6, 5 mMβ-mercaptoethanol and an NaCl gradient from 0-0.3 M (500-ml gradient;flow rate=2 ml/min).

After pooling and diluting the relevant fractions, the pH was adjustedto 8 with 1 M Tris-HCl, pH 8. Q-sepharose chromatography (PharmaciaQ-sepharose, fast flow; 16-ml bed volume column) was performed with 20mM Tris-HCl, pH 8, 5 mM β-mercaptoethanol and a NaCl gradient from 0-0.5M (120-ml gradient; flow rate=1 ml/min). The relevant fractions werepooled, concentrated, and then applied to a Pharmacia Superose 6 gelfiltration column (10 mM Tris-HCl, 1 mM EDTA, pH 8, 5 mMβ-mercaptoethanol; flow rate=0.5 ml/min), with the lactacystin-bindingactivity corresponding to a single high molecular weight peak. This peakwas isolated and treated with 10 μM [³H]lactacystin or 10 μM[³H]β-lactone for 24 h at 25° C. Trifluoroacetic acid (TFA) was added to0.1%, and the sample was allowed to stand at room temperature for 20min. Reverse-phase HPLC was then carried out at room temperature using aVydac C4 column (300 Å/4.6×150 mm) with 20-40% acetonitrile/0.1% TFAover 10 min and then 40-55% acetonitrile/0.1% TFA over 30 min (flowrate=0.8 ml/min). An IN/US β-RAM in-line scintillation detector was usedto monitor radioactivity.

The lactacystin-binding proteins were purified from bovine brain, andboth were found to reside in the same high-molecular weight proteincomplex by gel-filtration chromatography. Treatment of the complex with[³H]lactacystin did not cause its dissociation, and the radioactivityuniquely comigrated with the complex. The molecular weight of thecomplex was estimated to be 700 kDa, and SDS-PAGE revealed that itconsisted of numerous proteins with molecular weights of ˜24-32 kDa.Edman degradation of blotted protein revealed the sequences of severalproteasome subunits in the 24 kDa band, leading to a tentativeidentification of the complex as the 20S proteasome. After the complexwas treated with [³H]lactacystin and subjected to reverse-phase HPLC toseparate the proteasome subunits, eleven to twelve distinct peaks wereresolved. However, the radioactivity was associated exclusively with thefirst two peaks, and predominantly with the second. These first twopeaks were judged to be homogeneous by Coomassie blue staining ofSDS-polyacrylamide gels and by sequencing of tryptic fragments, whilesome of the subsequently eluting, larger peaks were clearly nothomogeneous. The ratio of counts incorporated into the first peak versusthe second peak varied with the batch of protein, the length ofincubation, and the ligand. The first peak is labeled more slowly, andthe degree of labeling of the first peak relative to the second isgreater with [³H]lactacystin than with the [³H]β-lactone at any giventime. A one or two hour reaction with [³H]β-lactone results in onlytrace labeling of the first peak, while a 24 hour reaction with[³H]β-lactone or [³H]lactacystin results in significant labeling of thispeak. The selectivity runs opposite to the relative chemical reactivityof the two compounds, and this finding suggests that theN-acetylcysteine moiety of lactacystin may facilitate recognition bythis secondary protein. The first peak to elute from the reverse-phaseHPLC column contained only a ˜32 kDa protein, which corresponded to the32 kDa secondary lactacystin-binding protein identified earlier.

Example 4

Amino Acid Sequence of Purified Bovine Lactacystin-binding Proteins

Purified 20S proteasome (purified as described in Example 3) wasincubated for 24 h at room temperature with 10 μM lactacystin in 10 mMTris-HCl, 1 mM EDTA (pH 8). The solution was then diluted tenfold with20% aqueous acetonitrile containing 0.1% trifluoroacetic acid (TFA) andallowed to stand at room temperature for 5 min. Reverse-phase HPLC wasthen performed at room temperature using a Vydac C4 column (100Å/4.6×150 mm) with 20-40% acetonitrile/0.1% TFA over ten minutes andthen 40-55% acetonitrile/0.1% TFA over 30 min to elute the proteasomesubunits (flow rate=0.8 ml/min). Peaks were collected based onultraviolet light absorbance at 210 and 280 nm, with the primarylactacystin-modified protein being the second to elute off the columnand the secondary lactacystin-binding protein being the first. Proteinfrom repeated injections was pooled, lyophilized and subjected to Edmandegradation following tryptic digestion and reverse-phase HPLCseparation of tryptic fragments. The putative lactacystin-modifiedresidue was identified by adding subunit X/MB1 isolated from[³H]lactacystin-treated proteasome to a sample that had been treatedwith unlabeled lactacystin, and then isolating and sequencingradioactive tryptic fragments. At one position, thephenylthiohydantoin-amino acid derivative was not identifiable,indicating possible modification of the residue.

Sequences from direct N-terminal sequencing and from tryptic fragmentsderived from the primary bovine lactacystin-binding protein aligned withsequences of human proteasome subunit X (predicted from the cDNA clone),and human LMP7-E2 (predicted from the exon 2-containing cDNA clone).Sequence 1 (from direct N-terminal sequencing) is also aligned withsequences of Saccharomyces cerevisiae Pre-2 (predicted from the genomicclone) and Thermoplasma acidophilum β-subunit (predicted from the clonedgene). The N-terminal heptapeptide corresponds to the tryptic fragmentthat appears to contain a lactacystin-modified N-terminal threonineresidue. The threonine at this position also corresponds to the putativeN-termini of the mature, processed forms of all the homologs listed.Upper-case letters denote high confidence sequence, while lower caseletters indicate lower confidence assignments.

TABLE 1 DIRECT N-TERMINAL SEQUENCES 1. Direct N-terminal sequence ofprimary bovine lactacystin-binding protein TTTLAFKFRHggIIA SEQ ID NO: 1Human subunit X/MB1 5 TTTLAFKFRHGVIVA 19 SEQ ID NO: 2 Human LMP7-E2 74TTTLAFKFQHGVIAA 88 SEQ ID NO: 3 S. cerevisiae Pre-2 76 TTTLAFRFQGGIIVA90 SEQ ID NO: 4 T. acidopholum β-Subunit 9 TTTVGITLKDAVIMA 23 SEQ ID NO:5 2. Primary bovine lactacystin-binding protein DAYSGGSVSLY SEQ ID NO: 6Human subunit X 171 DAYSGGAVNLYHVR 184 SEQ ID NO: 7 Human LMP7-E2 239DSYSGGVVNMYHMK 252 SEQ ID NO: 8 3. Primary bovine lactacystin-bindingprotein VIEINPYLLGTMAGGAADCSF SEQ ID NO: 9 Human subunit X 38VIEINPYLLGTLAGGAADCQFWER 61 SEQ ID NO: 10 Human LMP7-E2 106VIEINPYLLGTMSGCAADCQYWER 129 SEQ ID NO: 11 4. Primary bovinelactacystin-binding protein GYSYDLEVEEAYDLAR SEQ ID NO: 12 Human subunitX 146 GYSDLEVEQAYDLAR 161 SEQ ID NO: 13 Human LMP7-E2 214GYRPNLSPEEAYDLGR 229 SEQ ID NO: 14

Sequences of tryptic fragments derived from the secondarylactacystin-binding protein aligned with N-terminal fragment sequence ofhuman erythrocyteproteasome a chain and of rat liver proteasome chain.

TABLE 2 TRYPTIC FRAGMENT SEQUENCES Secondary bovine lactacystin-bindingactivity TTIAGVVYK DGIVLGADTR SEQ ID NO: 15 Human erythrocyte proteasomeαchain 1 XXIAGVVYK DGZVLGADTR 19 SEQ ID NO: 16 Rat liver proteasomechain 1 1 TTIAGVVYK DGI 12 SEQ ID NO: 17

Sequence analysis of tryptic fragments derived from this protein showedit to be homologous to the proteasome a chain, a ˜30 kDa proteinidentified in purified human erythrocyte proteasome and rat liverproteasome for which only an N-terminal fragment sequence exists. Thesecond peak to elute contained only a ˜24 kDa protein, the primarylactacystin-binding protein. Sequence from the N-terminus of the proteinand from derived tryptic fragments showed high identity to the recentlydiscovered 20S proteasome subunit X, also known as MB1, a homolog of themajor histocompatibility complex (MHC)-encoded LMP7 proteasome subunit.

Example 5

Kinetics of Inhibition of 20S Proteasome Peptidase Activities

Experiments involving the proteasome were performed as follows: 20Sproteasome (˜5 ng/μl) in 10 mM Tris-HCl, pH 7.5, 1 mM EDTA was incubatedat 25° C. in the presence of lactacystin or lactacystin analogs in DMSOor MeOH [not exceeding 5% (v/v)]. Aliquots for fluorescence assay wereremoved at various times following addition of compound and diluted in10 mM Tris-HCl, pH 7.5, 1 mM EDTA containing fluorogenic peptidesubstrates (100 μM final). Suc-LLVY-AMC, Cbz-GGR-βNA and Cbz-LLE-βA(AMC=7-amido-4-methylcoumarin; βNA=β-naphthylamide) were used toassayfor the chymotrypsin-like, trypsin-like and peptidylglutamyl-peptidehydrolyzing activities, respectively.

Biological activity of compound refers to the ability of the compound toinduce neurite outgrowth in Neuro 2A neuroblastoma cells and to inhibitcell cycle progression in MG-63 osteosarcoma cells.

TABLE 3 Kinetics of Inhibition κ_(assoc) = κ_(obs)/[I](s⁻¹M⁻¹)Biological Chymotrypsin-like Peptidylglutamyl- Compound activity ofactivity (Suc- Trypsin-like activity peptide hydrolyzing (concentration)compound LLVY-AMC) (Coz-GGR-βNA) activity(Cbz-LLE-βNA) Lactacystin (10μM) + 194 ± 15 10.1 ± 1.8 Lactacystin (100 μM) + 4.2 ± 0.6 β-Lactone (1μM) + 3059 ± 478 β-Lactone (5 μM) + 208 ± 21 β-Lactone (50 μM) + 59 ± 17Dihydroxy acid (100 μM) − Na inhibition No inhibition No inhibitionLactacystin amide (12.5 μM) + 306 ± 99 Phenylacetyl lactacystin + 179 ±19 (12.5 μM) 6-Deoxylactacystin − No inhibition (12.5 μM) (6R,7S)Lactacystin (6-epi, 7- − No inhibition epi)(12.5 μM)Des(hydroxyisobutyl)-lactacy − No inhibition stin (12.5 μM)

Example 6

Selectivity of Protease Inhibition

Experiments involving the other proteases were similar to Example 5,except that buffers used forincubation with lactacystin were as follows:α-Chymotrypsin: 10 mM Tris-HCL, pH 8, 1 mM EDTA (plus 100 μMSuc-LLVY-AMC for fluorescence assay); Trypsin: 10 mM Tris-HCL, pH 8, 20mM CaCl₂ (plus 100 μM Cbz-GGR-βNA for assay); Calpain I: 20 mM Tris-HCL,pH 8, 1 mM CaCl₂, 1 mM DTT (plus 100 μM Suc-LLVY-AMC for assay); CalpainII: 20 mM Tris-HCL, pH 8, 10 mM CaCl₂, 1 mM DTT (plus 100 μMSuc-LLVY-AMC for assay); Papain: 50 mM MES—NaOH, pH 6.4, 1 mM DTT (plus100 μM Cbz-RR-AMC for assay); Cathepsin B: 100 mM KH₂PO₄, pH 5.5, 2 mMEDTA, 1 mM DTT (plus 100 μM Cbz-RR-AMC for assay). Fluorescent emissionat 460 nm with excitation at 380 nm was measured for AMC-containingsubstrates, and emission at 410 nm with excitation at 335 nm wasmeasured for RNA-containing substrates. The fluorescence assays wereconducted at 25° C., each experiment was performed at least three times,and values represent mean±standard deviation.

TABLE 4 Inhibition of Other Proteases Effect of lactacystin Proteasetested (100 μM) α-Chymotrypsin No inhibition Trypsin No inhibitionCalpain I No inhibition Calpain II No inhibition Papain No inhibitionCathepsin B No inhibition

The effects of lactacystin and the β-lactone on proteasame peptitase onactivities using fluorogenic peptide substrates were assessed. All threepeptidase activities were inhibited, irreversibly in the case of thetrypsin-like and chymotrypsin-like activities and reversibly in the caseof the PGPH activity. The apparent second-order rate constant forassociation of each inhibitor with the enzyme, k_(assaoc), wasdetermined for each of the activities (Table 1A). Lactacystin inhibitsthe chymotrypsin-like activity the fastest (k_(assoc)=194±15 M-1_(S)-1),the trypsin-like activity 20-fold more slowly, and the PGPH activity50-fold more slowly. The fact that the inhibition kinetics are differentfor the three activities lends further support to the notion that theactivities are due to separate active sites. The β-lactone displays thesame rank order but inhibits each activity 15-20 times faster than doeslactacystin itself, in accord with the greater expected chemicalreactivity of the β-lactone. It is also possible that, upon initialbinding to the protein target, the lactacystin thioester is cyclized ina rate-limiting step to the β-lactone, which serves as an activatedintermediate for attack by the nucleophile.

The reversibility of the inhibitory effects was assessed by measuringresidual peptidase activity after removal of excess inhibitor byextensive serial dilution/ultrafiltration. The trypsin-like andchymotrypsin-like activities were still completely inhibited in thelactacystin-treated samples following dilution/ultrafiltration, implyingvery low k_(off) of inhibitor from enzyme, whereas controls untreatedwith inhibitor maintained activity (data not shown). In the case of thePGPH activity, removal of the inhibitor was accompanied by a return ofthe catalytic activity; inhibition of the PGPH activity could be due tonon-covalent association of lactacystin with the PGPH site orcovalentassociation with turnover.

Previously, we had shown that the ability of analogs to cause neuriteoutgrowth in Neuro 2A cells was mirrored by their ability to inhibitcell-cycle progression in MG-63 osteosarcoma cells, and thatmodifications to the γ-lactam part of the molecule impared bothactivities whereas modifications of the N-acetylcysteine moiety hadlittle effect [Fenteany, 1994 3135]. We therefore tested the ability ofthe analogs to inhibit the 20S proteasome, but as our supplies of thesematerials were insufficient to examine all three activities, we focusedon the chymotrypsin-like activity, which is inhibited most rapidly bylactacystin. The same trends found in the biological studies wereapparent: the biologically active compounds inhibited thechymotryptsin-like activity about as well as lactacystin itself, and thebiologically inactive compounds did not inhibit this activity as all(Table 3). Modifications of the γ-lactam core of lactacystin mitigateits effect, but modifications of the N-acetylcysteine moiety do not.

Example 7

Assay of the Ability of Lactacystin (β-lactone Form) can Block TNF-αDependent Degradation of IκB-α in vivo.

Hela cells were plated onto 6-well plates in DME plus 10% fetal calfserum (3 mls/well). Cells were then pretreated with 0.125% DMSO 40 μMG132 (carbobenzoxyl-leucinyl-leucinyl-leucinal-H)(40 μM, lanes 103), or5 μM β-lactone for one hour, followed by treatment with 1,000 U/ml TNF-αor phosphate buffered saline (PBS). Cells were harvested after 0 min, 20min, or 40 min of further incubation at 37° C. Cells were then lysed inbuffer containing NP-40 and protease inhibitors, and the proteinsseparated on a 10% SDS-polyacrylamide gel. The proteins were thentransferred to nitrocellulose and probed with antibodies against theC-terminal 20 amino acids of human IκB-α.

Previous reports have shown that the inducible degradation of IκB ispreceded by phosphorylation of the protein (Beg et al., 1993, Mol. Cell.Biol. 13:3301; Traenckner et al., 1994, EMBO J. 13:5433; Miyamoto etal., 1994, PNAS 91:12740; Lin et al., 1995, PNAS 92:552; DiDonato etal., 1995, Mol. Cell. Biol. 15:1302; and Alkalay et al., 1995, Mol.Cell. Biol. 15:1294). The phosphorylated IκB is then preferentiallydegraded, apparently by the proteasome (Palombella et al., 1994, Cell78:773). Phosphorylation is evidenced by a slightly heavier shift in theelectrophoretic mobility of IκB. IκB from cells induced with TNF showthe requisite pattern of phosphorylation and degradation over time.Cells treated with PBS only show no change in the level or modificationof IκB. As was reported previously, (Palombella et al, 1994), thepeptide aldehyde proteasome inhibitor MG132 blocks the degradation ofIκB and stabilizes the phosphorylated form. β-lactone also blocksdegradation and stabilizes the phosphorylated form of IκB. These resultsindicate that lactacystin does not effect the TNF dependent signallingpathway that leads to the phosphorylation of IκB, but does block thedegradation of the protein. Lactacystin inhibits the degradation of IκBafter TNF induction of Hela cells, most likely by specifically blockingthe action of the proteasome.

Example 8

Assay of the Effect of Lactacystin (β-lactone form) on the ProteasomeDependent p105 to p50 Processing in vivo.

COS cells were plated onto 100 mm dishes, then, with DEAE-Destran,either mock transfected, or transfected with 3 μg of pcDNA plasmidcontaining the human p105 cDNA. Forty-eight hrs after transfection,cells were pretreated for 1 hour with 0.5% DMSO, 50 μM calpain inhibitorII, 50 μM MG132, or 10 μM β-lactone. Cells were then pulse labelled with250 uCi/plate of ³⁵S-methionine/cysteine for 15 minutes, and eitherharvested immediately or followed by a 2 hour chase with excessunlabelled methionine and cysteine. Cells were then lysed with SDS/Tris,and proteins were immunoprecipitated with anti-p50 antibodies (recognizethe N-terminal half of the p105 protein). These proteins were thenseparated on a 10% SDS-polyacrylamide gel, which was then fixed, dried,and exposed to autoradiographic film.

An analysis of the proteins isolated immediately after thepulse-labelling period reveals significant amounts of labelled p105protein and very little p50. After the 2 hour chase period, the levelsof p105 were reduced, and a new band that corresponds to p50 protein isapparent, as was expected (Fan and Maniatis, 1991, Nature 354:395;Palombella et al., 1994, Cell 78:773). Pretreatment of cells withcalpain inhibitor II has no effect on the processing of p105 to p50(lane 4). However, treatment of cells with the peptide aldehydeproteasome inhibitor MG132 completely blocks the appearance of p50 (lane5), as has been reported previously (Palombella et al., 1994). Lowlevels of β-lactone (10 μM) have only a slight effect on the levelofp50, but higher concentrations (50 μM) result in the completeinhibition of the processing of p105 to p50. Lactacystin efficientlyblocks the proteasome dependent procession of p105 to p50 in vivo.

Example 9

Neurite Outgrowth Assay

Compounds are dissolved in the minimal amount of methanol (MeOH) ordimethyl sulfoxide (DMSO) required for solubilization. No more than 0.1%solvent is present in any assay. When necessary, solutions areevaporated to dryness and resuspended in cell culture medium to theirfinal concentrations before use.

Neuro 2A, IMR-32, PC12, and MG-63 cells are obtained from the AmericanType Culture Collection. Neuro 2A and IMR-32 cells are cultured inEagle's minimal essential medium (MEM) containing 10% (vol/vol) fetalbovine serum (FBS). PC12 cells are grown in RPMI 1640 medium containing10% (vol/vol) horse serum and 5% FBS, and MG-63 cells are cultured inRPMI 1640 containing 10% FBS.

Neuro 2A cells are plated at a density of 1×10⁴ cells per 1 ml per wellin 12-well polystyrene dishes (22-mm-diameter flat-bottom wells) and aregrown for 24 h in MEM with 10% FBS prior to any treatment. In therelevant experiments, nocodazole, cytochalasin B, or cycloheximide isadded 3 h before addition of lactacystin. In the serum deprivationexperiments, cells are switched to serum-free MEM 24 h after platingand, when relevant, incubated another 24 h before addition oflactacystin and subsequently maintained in serum-free conditions.

Example 10

Cell Cycle Analysis

MG-63 cells are plated at 7.5×10⁴ cells per 3 ml per 25-cm² flask andgrown for 24 h in RPMI containing 10% FBS. These subconfluent MG-63cultures are synchronized in G₀/G₁ by changing the medium to RPMIcontaining 0.2% FBS and incubating for 64 h. This is followed bystimulation with 2 ml of RPMI containing 10% FBS and addition ofcompounds. Neuro 2A cells are grown to ˜2×10⁷ in 175-cm² flasks in MEMwith 10% FBS. Mitotic cells are harvested by shaking for 5 min at 100rpm on a rotary shaker. The detached cells are replated at 1.5×10⁵ cellsper 2 ml per 25-cm² flask and incubated for 30 min to allow forreattachment prior to addition of lactacystin. Cells are harvested forcell cycle analysis 21 h after stimulation in the case of the MG-63cells and 20 h after replating in the case of the Neuro 2A cells andthen were processed for flow cytometry. DNA histograms are obtainedusing a Becton Dickinson FACScan flow cytometer.

Example 11

Purification of 20S Proteasome and Proteasome Activator PA28

20S proteasome was purified by a modification of procedures by Hough etal. [Hough, R., et al., (1987) J. Biol. Chem., 262, 8303-8313] andGanoth et al. [Ganoth, D., et al., (1988) J. Biol. Chem., 263,12412-12419. PA28 Activator was purified by a modification of proceduresof Chu-Ping et al. [Chu-Ping, M., et al., (1992) J. Biol. Chem., 267,10515-10523]. Rabbit reticulocytes were obtained from Green Hectares(Oregon, Wis.). The reticulocytes were washed three times by suspendingthem in ice cold PBS and centrifuging at 2000×g for 15 min. After thefinal wash, the cells were lysed in 1.5 volumes of cold distilled watercontaining 1 mM DTT. The mixture was then passed through a Glas-COBio-Nebulizer at 250 psi to ensure complete cell lysis. The lysate wasthen centrifuged at 100,000×g for one hour to remove cell debris. Thesupernatant (extract) was brought to 20 mM HEPES pH 7.6, 1 mM DTT(Buffer A), filtered through a 0.2 μm filter, and applied to a DE52anion exchange column equilibrated in Buffer A. The column was washedwith 5 volumes of Buffer A and absorbed proteins were eluted with 2.5volumes of Buffer A containing 500 mM NaCl. The eluate (Fraction II) wasbrought to 38% saturation with ammonium sulfate and centrifuged at10,000×g for 20 minutes. The supernatant was brought to 85% saturationwith ammonium sulfate and centrifuged 10,000×g for 20 minutes. Theresulting pellets were resuspended in a minimal volume of Buffer A anddialyzed vs. 4L of Buffer A overnight. The dialysate (Fraction IIB),which contains both 20S proteasome and PA28 activator, was applied to aMono Q anion exchange column. Elution was performed by a 40 columnvolume gradient from 0-500 mM NaCl.

20S Proteasome

Column fractions were assayed for SDS-stimulated Suc-Leu-Leu-Val-Tyr-AMChydrolyzing activity. The active fractions were pooled, diluted to 50 mM[NaCl], and applied to a 1 mL Heparin Sephrose Hi-Trap column. Elutionwas performed with a 20 column volume gradient from 50 mM-500 mM NaCl.Active fractions were pooled and concentrated using a 30,000 MWCOCentricon centrifugal concentrator. The concentrate was applied to aSuperose 6 size exclusion chromatography column and eluted with Buffer Acontaining 100 mM NaCl. The active fractions that were judged to be pureby SDS-PAGE analysis were pooled and stored at −80° C. for subsequentuse.

PA28

Column fractions (vide supra) were assayed for their ability tostimulate the Suc-Leu-Leu-Val-Tyr-AMC hydrolysing activity of exogenous20S proteasome. The active fractions were pooled and diluted to 10 mMNaCl. This material was then further purified and concentrated on a 1 mlResource Q anion exchange column with a 40 column volume gradient from10-500 mM NaCl. Active fractions were pooled and concentrated using a10,000 MWCO Centricon centrifugal concentrator. The concentrate wasapplied to a Superdex 200 size exclusion chromatography column andeluted with Buffer A containing 100 mM NaCl. The active fractions thatwere judged to be pure by SDS-PAGE analysis were pooled and stored at−80° C. for subsequent use.

Example 12

Lactacystin Inactivation of Proteasome Activity

2 ml of assay buffer (20 mM HEPES, 0.5 mM EDTA, pH 8.0) andSuc-Leu-Leu-Val-Tyr-AMC in DMSO were added to a 3 ml fluorescent cuvetteand the cuvette was placed in the jacketed cell holder of a HitachiF-2000 fluorescence spectrophotometer. The temperature was maintained at37° C. by a circulating water bath. 0.34 μg of proteasome and 3.5 μg ofPA28 were added and the reaction progress was monitored by the increasein fluorescence at 440 nm (λex=380 nm) that accompanies production offree AMC. The progress curves exhibited a lag-phase lasting 1-2 minutesresulting from the slow formation of the 20S-PA28 complex. Afterreaching a steady-state of substrate hydrolysis, lactacystin was addedto a final concentration of 1 μM and the reaction was monitored for 1hr. The fluorescence (F) versus time (t) data were collected on amicrocomputer using LAB CALC (Galactic) software. k_(inact) values wereestimated by a nonlinear least-squares fit of the data to thefirst-order equation:

F=A(1−e ^(−kt))+C

where C=F_(t=0) and A=F_(t∞)−F_(t=0). Owing to the complex kinetics oflactacystin hydrolysis and its mechanism of inactivation of theproteasome the kinetics of proteasome inactivation are also complex andthis fact is reflected in the curve fitting. There was a smallsystematic deviation of the measured progress curves from the “best-fit”curves generated by fitting the data to the equation above. Estimates ofk_(inact) were obtained using this simple kinetic model.

Example 13

Inhibition of Protein Degradation in C2C12 Cells

C2C12 cells (a mouse myoblast line) were labeled for 48 hrs with³⁵S-methionine. The cells were then washed and preincubated for 2 hrs inthe same media supplemented with 2 mM unlabeled methionine. The mediawas removed and replaced with a fresh aliquot of the preincubation mediacontaining 50% serum, and a concentration of the compound to be tested.The media was then removed and made up to 10% TCA and centrifuged. TheTCA soluble radioactivity was counted. Inhibition of proteolysis wascalculated as the percent decrease in TCA soluble radioactivity. Fromthis data, an IC₅₀ for each compound was calculated. The compounds canbe qualitatively grouped into ++, +, and 0 categories, wherein wherein++ indicates better activity than +, and 0 indicates little or noactivity, due to insolubility, instability, or other reasons. Forexample, in the lactacystin group compounds I-1, I-2, and I-3 were inthe ++ category; compound I-4 was in the + category. Turningto the Pseries of lactone-like compounds, P-1 through P-4 were in the ++category; compounds P-5 and P-6 were in the + category; and compound P-7was in the 0 category.

Example 14

Synthesis of Compound I-1

(3R,4S, 5R,1′S)-4-Hydroxy-5(1′-hydroxy-2′-methylpropyl)-3-methylpyrrolidin-2-one-5-carboxylicacid (10.0 mg, 43.2 mmol; made by the route described by Corey, E. J.;Reichard, G. A., J. Am. Chem. Soc. 1992, 114, 10677) was dissolved in0.4 mL anhydrous dichloromethane and then treated, in order, withtri-ethylamine (18.0 mL, 0.13 mmol, 3.0 equiv.), 13.0 mgbis(2-oxo-3-oxazolidinyl)-phosphinic chloride (51.1 mmol, 1.2 equiv.)and benzyl mercaptan (8.0 mL, 68.1 mmol, 1.58 equiv.). The solution wasstirred for 5 hours at room temperature and then diluted withdichloromethane and washed with water. The aqueous layer was extractedwith ethyl acetate and the organic layers were combined and dried overmagnesium sulfate. Filtration followed by removal of the solventafforded a crude solid which was triturated with 19:1 hexane/ethylacetate and collected by filtration. The white solid obtained was thendissolved in a minimum amount of acetonitrile and the clear solution wasdiluted with water then frozen and lyophilized overnight. This afforded8.0 mg of the desired thioester I-1 (55%) as a fluffly white solid. ¹HNMR (DMSO-d₆, 300 MHz) d 8.34 (s, 1H, NH), 7.19-7.28 (m, 5H), 5.40 (d,1H, ³J=6.4 Hz, OH), 5.11 (d, 1H, ³J=7.4 Hz, OH), 4.39 (t, ³J=6.4 Hz),4.04 (s, 2H), 3.76 (t, 1H, ³J=7.4 Hz), 2.62 (m, 1H), 1.55 (m, 1H), 0.90(d, 3H, ³J=7.5 Hz), 0.85 (d, 3H, ³J=6.6 Hz), 0.66 (d, 3H, ³J=6.8 Hz).

Example 15

Synthesis of Compound I-2

To a solution of glutathione (360 mg, 1.17 mmol) in 11 mL of water wasadded 1.35 mL of 1.004 N sodium hydroxide in water. This solution wasthen added to a freshly prepared solution ofclasto-lactacystin-b-lactone (49.0 mg, 0.23 mmol; made by the routedescribed by Corey, E. J.; Reichard, G. A. J. Am. Chem. Soc. 1992, 114,10677) in 10 mL of acetonitrile. After stirring the solution at roomtemperature for 4 hours, the pH was adjusted to about pH=4 by the slowaddition of 1 N aqueous hydrochloric acid solution. The acetonitrile wasremoved in vacuo and the aqueous phase was frozen and lyophilized. Thesolid obtained was redissolved in water, filtered and lyophilized again.The white solid obtained was purified by reverse phase HPLC [Waters PrepDelta-Pak® 19×300 mm C18 column eluting with 98% 0.05% trifluoroaceticacid/H₂O and 2% 0.05% trifluoroacetic acid/methanol to 80% 0.05%trifluoroacetic acid/H₂O and 20% 0.05% trifluoroacetic acid/methanol in65 min at 5 mL/min]. After lyophilizing the pure fractions, glutathionethioester adduct I-2 (48 mg, 40%) was obtained as a fluffly white solid.¹H NMR (DMSO-d6, 300 MHz) d 7.73-8.26 (m, 4H), 5.28 (bs, 1H, OH), 5.09(bs, 1H, OH), 4.44 (m, 1H), 4.37 (bd, 1H, 3J=5.9 Hz), 3.74 (m, 2H), 3.23(dd, 1H, J=13.3, 4.6 Hz), 2.95 (dd, 1H, J=13.3, 8.8 Hz), 2.62 (m, 1H),2.29-2.32 (m, 2H), 1.98-2.08 (m, 2H), 1.51-1.63 (m, 1H), 0.92 (d, 3H,³J=7.5 Hz), 0.86 (d, 3H, 3J=6.6 Hz), 0.71 (d, 3H, ³J=6.7 Hz).

Example 16

Synthesis of Compound I-4

(3R,4S,5R,1′S)-4-Hydroxy-5(1′-hydroxy-2′-methylpropyl)-3-methylpyrrolidin-2-one-5-carboxylicacid (20.0 mg, 86.5 mmol; made by the route described by Corey, E. J.;Reichard, G. A. J. Am. Chem. Soc. 1992, 114, 10677) was dissolved in 1.0mL acetonitrile and then treated, at 0° C., with1,8-diazabicyclo[5.4.0]undec-7-ene (14.0 mL, 93.6 mmol, 1.08 equiv.)followed by benzyl bromide (12.0 mL, 101 mmol, 1.17 equiv.). The mixturewas stirred at room temperature overnight and was then partitionedbetween ethyl acetate and water. The aqueous layer was extracted threetimes with ethyl acetate and the organic layers were then combined anddried over magnesium sulfate. Filtration followed by removal of thesolvent afforded a crude solid which was triturated with hexane andcollected by filtration. The white solid obtained was then dissolved ina minimum amount of acetonitrile and the clear solution was diluted withwater then frozen and lyophilized over-night. This afforded 6.7 mg ofthe desired ester I-4 (24%) as a fluffly white solid. ¹H NMR (DMSO-d₆,300 MHz) d 8.02 (s, 1H, NH), 7.31-7.44 (m, 5H), 5.45 (d, 1H, 3J=6.4 Hz,OH), 5.11 (d, 1H, 2J=12.5 Hz), 5.03 (d, 1H, 3J=7.2 Hz, OH), 5.00 (d, 1H,2J=12.5 Hz), 4.31 (t, 3J=6.4 Hz), 3.76 (t, 1H, ³J=7.2 Hz), 2.68 (m, 1H),1.52 (m, 1H), 0.89 (d, 3H, 3J=7.4 Hz), 0.85 (d, 3H, 3J 6.6 Hz), 0.67 (d,3H, 3J=6.7 Hz).

Example 17

Synthesis of Compound P-2

(3S,7aR)-3-Phenyl-5,6,7,7a-tetrahydro-#1H-pyrrolo[1,2-c]oxazol-5-one wasprepared in three steps from D-Pyroglutamic acid by a known procedure(Thottathil, J. K. et al. J. Org. Chem. 1986, 51, 3140) and wasconverted to (3S,7aR)-3-Phenyl-1H-pyrrolo[1,2-c]oxazol-5-one in twosteps (selenylation and oxidative elimination) by the method reported byHamada et al. (J. Am. Chem. Soc. 1989, 171, 1524).(3S,7aR)-3-Phenyl-1H-pyrrolo[1,2-c]oxazol-5-one was then converted to(3S,7S,7aS,1′S)-7a-(1′-acetoxy-2′-methylpropyl)-7-hydroxy-3-phenyl-5,6,7,7a-tetrahydro-1H-pyrrolo[1,2-c]oxazol-5-onein 6 steps by analogy to the sequence reported by Uno et al. (J. Am.Chem. Soc. 1994, 116, 2139).(3S,7S,7aS,1′S)-7a-(1′-acetoxy-2′-methylpropyl)-7-hydroxy-3-phenyl-5,6,7,7a-tetrahydro-1H-pyrrolo[1,2-c]oxazol-5-one(70 mg, 0.21 mmol) was dissolved in 1.0 mL pyridine and treated at roomtemperature with 32.0 mL acetic anhydride (0.34 mmol, 1.6 equiv.). Themixture was stirred at room temperature overnight and the solvent wasremoved in vacuo affording 77 mg of desired(3S,7S,7aS,1′S)-7a-(1′-acetoxy-2′-methylpropyl)-7-acetoxy-3-phenyl-5,6,7,7a-tetrahydro-1H-pyrrolo[1,2-c]oxazol-5-one(97%), a yellow oil which was used as such in the followingstep.

The crude(3S,7S,7aS,1′S)-7a-(1′-acetoxy-2′-methylpropyl)-7-acetoxy-3-phenyl-5,6,7,7a-tetrahydro-1H-pyrrolo(1,2-c]oxazol-5-one(75 mg, 0.20 mmol) was dissolved in 5 mL methanol and placed under 1atmosphere of hydrogen in the presence of 50 mg 20% palladium hydroxideon carbon. When a TLC analysis (1:1 hexane/ethyl acetate) showed thereaction to be complete, the mixture was filtered and concentrated invacuo affording 55 mg (97%) of the desired primary alcohol which withoutany purification was converted to(4S,5R,1′S)-4-Hydroxy-5-(1′-hydroxy-2′-methylpropyl)-pyrrolidin-2-one-5-carboxylicby a two step sequence (Jones' oxidation, saponification) analogous tothe sequence reported by Uno et al. (J. Am. Chem. Soc. 1994, 116, 2139).(4S,5R,1′S)-4-Hydroxy-5-(1′-hydroxy-2′-methylpropyl)-pyrrolidin-2-one-5-carboxylicacid (7.0 mg, 32 mmol) was dissolved in 2.0 mL 1:1acetonitrile/tetrahydrofuran and then treated, at room temperature, with2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyl uronium tetrafluoroborate(11.0 mg, 34 mmol, 1.1 equiv.) followed by triethylamine (0.36 mmol, 11equiv.). The mixture was stirred for 30 min to room temperature andconcentrated in vacuo. Flash chromatography (230-400 mesh SiO, elutionwith ethyl acetate) finally afforded 3 mg of the pure lactone P-2 (47%)obtained as white solid. H NMR (DMSO-d6, 300 MHz) d 8.95 (bs, 1H, NH),5.64 (d, 1H, 3J=6.7 Hz, OH), 5.26 (d, 1H, 3J=6.0 Hz, OH), 3.78 (dd, 1H,3J=6.7, 3.5 Hz), 2.81 (dd, 1H, 2J=19.2 Hz, 3J=6.0 Hz), 2.60 (d, 1H,2J=19.2 Hz), 1.74 (m, 1H), 0.90 (d, 3H, 3J=6.9 Hz), 0.68 (d, 3H, ³J=6.8Hz).

Example 18

Synthesis of Compound P-3

Clasto-Lactacystin-β-lactone (5.0 mg, 23.4 mmol; made by the routedescribed by Corey, E. J.; Reichard, G. A. J. Am. Chem. Soc. 1992, 114,10677) was dissolved in 1.5 mL pyridine and treated, at room temperaturewith acetic anhydride (40 mL, 0.42 mmol, 18 equiv.). After stirring thesolution at room temperature overnight, the pyridine was removed invacuo with the aid of 2 mL of toluene affording a light brown solid. Thesolid was washed with three portions of hexane and then filtered giving5.5 mg of the pure acetate P-3 (93%) obtained as a white solid. ¹H NMR(DMSO-d₆, 300 MHz) d 9.11 (bs, 1H, NH), 5.33 (d, 1H, ³J=6.1 Hz), 5.21(d, 1H, ³J=5.0 Hz), 2.84 (m, 1H), 2.06 (s, 3H), 1.90 (m, 1H), 1.08 (d,3H, ³J=7.5 Hz), 0.87 (d, 3H, ³J=6.9 Hz), 0.78 (d, 3H, ³J=6.8 Hz).

Example 19

Synthesis of Compound P-4

(3S, 7aR)-3-Phenyl-5,6, 7, 7a-tetrahydro-1H-pyrrolo[1,2-c]oxazol-5-onewas prepared in three steps from #D-Pyroglutamic acid by a knowprocedure (Thottathil, J. K. et al. J. Org. Chem. 1986, 51, 3140) andwas converted to (3S,7aR)-3-Phenyl-1H-pyrrolo[1,2-c]oxazol-5-one in twosteps (selenylation, and oxidative elimination) by the method reportedby Hamada et al. (J. Am. Chem. Soc. 1989, 111, 1524).(3S,7aR)-3-Phenyl-1H-pyrrolo[1,2-c]oxazol-5-one was then converted to(3S,6R,7S,7aS,1′S)-7a-(1′-acetoxy-2′-methylpropyl)-6-hydroxy-7-hydroxy-3-phenyl-5,6,7,7a-tetrahydro-1H-pyrrolo[1,2-c]oxazol-5-onein 4 steps by analogy to the sequence reported by Uno et al. (J. Am.Chem. Soc. 1994, 116, 2139).

(3S,6R,7S,7aS,1′S)-7a-(1′-acetoxy-2′-methylpropyl)-6-hydroxy-7-hydroxy-3-phenyl-5,6,7,7a-tetrahydro-1H-pyrrolo[1,2-c]oxazol-5-onewas then converted in 4 steps (acetylation, hydrogenolysis, oxidationand saponification)to(3R,4S,5R,1′S)-4-Hydroxy-5-(1′-hydroxy-2′-methylpropyl)-3-hydroxypyrrolidin-2-one-5-carboxylicacid in the same manner described for making P-2.

(3R,4S,5R,1′S)-4-Hydroxy-5-(1′-hydroxy-2′-methylpropyl)-3-hydroxypyrrolidin-2-one-5-carboxylicacid (20 mg, 86 mmol) was converted to the corresponding β-lactone bythe method described for making P-2. This afforded 3 mg of the purelactone P-4 (16%) obtained as white solid. H NMR (DMSO-d₆, 300 MHz) d8.96 (s, 1H, NH), 6.08 (bs, 1H, OH), 5.63 (d, 1H, ³J=6.6 Hz, OH), 5.12(d, 1H, ³J=5.7 Hz), 4.31 (d, 1H, ³J=5.7 Hz), 3.72 (bt, 1H), 1.70 (m,1H), 0.88 (d, 3H, ³J=6.9 Hz), 0.72 (d, 3H, ³J=6.8 Hz).

Example 20

Synthesis of Compound P-6

Clasto-Lactacystin-β-lactone (11.8 mg, 55.3 mmol; made by the routedescribed by Corey, E. J.; Reichard, G. A. J. Am. Chem. Soc. 1992, 114,10677) was dissolved in 0.4 mL freshly distilled pyridine and treated,at room temperature with methanesulfonyl chloride (6.0 mL, 77.5 mmol,1.4 equiv.). The solution was stirred for 2 hours at room temperature,treated with more methanesulfonyl chloride (10.0 mL, 129 mmol, 2.3equiv.) and stirred at room temperature overnight. The dark solution wasthen concentrated in vacuo with 3×2 mL of toluene affording a yellowsolid. Flash chromatography (230-400 mesh SiO₂, elution with 1:1hexane/ethyl acetate) finally afforded 12.6 mg of the pure mesylate P-6(78%) obtained as fluffy white solid. ¹H NMR (CDCl₃, 300 MHz) d 6.28(bs, 1H, NH), 5.27 (d, 1H, ³J=6.1 Hz), 5.06 (d, 1H, ³J=4.0 Hz), 3.11 (s,3H), 2.80-2.89 (m, 1H), 2.24-2.34 (m, 1H), 1.33 (d, 3H, ³J=7.5 Hz), 1.14(d, 3H, ³J=7.0 Hz), 1.05 (d, 3H, ³J=6.8 Hz).

OTHER EMBODIMENTS

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, other embodiments are also within the claims.

17 15 amino acids amino acid Not Relevant linear protein 1 Thr Thr ThrLeu Ala Phe Lys Phe Arg His Gly Gly Ile Ile Ala 1 5 10 15 15 amino acidsamino acid Not Relevant linear protein 2 Thr Thr Thr Leu Ala Phe Lys PheArg His Gly Val Ile Val Ala 1 5 10 15 15 amino acids amino acid NotRelevant linear protein 3 Thr Thr Thr Leu Ala Phe Lys Phe Gln His GlyVal Ile Ala Ala 1 5 10 15 15 amino acids amino acid Not Relevant linearprotein 4 Thr Thr Thr Leu Ala Phe Arg Phe Gln Gly Gly Ile Ile Val Ala 15 10 15 15 amino acids amino acid Not Relevant linear protein 5 Thr ThrThr Val Gly Ile Thr Leu Lys Asp Ala Val Ile Met Ala 1 5 10 15 11 aminoacids amino acid Not Relevant linear protein 6 Asp Ala Tyr Ser Gly GlySer Val Ser Leu Tyr 1 5 10 14 amino acids amino acid Not Relevant linearprotein 7 Asp Ala Tyr Ser Gly Gly Ala Val Asn Leu Tyr His Val Arg 1 5 1014 amino acids amino acid Not Relevant linear protein 8 Asp Ser Tyr SerGly Gly Val Val Asn Met Tyr His Met Lys 1 5 10 21 amino acids amino acidNot Relevant linear protein 9 Val Ile Glu Ile Asn Pro Tyr Leu Leu GlyThr Met Ala Gly Gly Ala 1 5 10 15 Ala Asp Cys Ser Phe 20 24 amino acidsamino acid Not Relevant linear protein 10 Val Ile Glu Ile Asn Pro TyrLeu Leu Gly Thr Leu Ala Gly Gly Ala 1 5 10 15 Ala Asp Cys Gln Phe TrpGlu Arg 20 25 amino acids amino acid Not Relevant linear protein 11 ValIle Glu Ile Asn Pro Pro Tyr Leu Leu Gly Thr Met Ser Gly Cys 1 5 10 15Ala Ala Asp Cys Gln Tyr Trp Glu Arg 20 25 16 amino acids amino acid NotRelevant linear protein 12 Gly Tyr Ser Tyr Asp Leu Glu Val Glu Glu AlaTyr Asp Leu Ala Arg 1 5 10 15 15 amino acids amino acid Not Relevantlinear protein 13 Gly Tyr Ser Asp Leu Glu Val Glu Gln Ala Tyr Asp LeuAla Arg 1 5 10 15 16 amino acids amino acid Not Relevant linear protein14 Gly Tyr Arg Pro Asn Leu Ser Pro Glu Glu Ala Tyr Asp Leu Gly Arg 1 510 15 19 amino acids amino acid Not Relevant linear protein 15 Thr ThrIle Ala Gly Val Val Tyr Lys Asp Gly Ile Val Leu Gly Ala 1 5 10 15 AspThr Arg 19 amino acids amino acid Not Relevant linear protein 16 Xaa XaaIle Ala Gly Val Val Tyr Lys Asp Gly Ile Val Leu Gly Ala 1 5 10 15 AspThr Arg 9 amino acids amino acid Not Relevant linear protein 17 Thr ThrIle Ala Gly Val Val Tyr Lys 1 5

What is claimed is:
 1. A pharmaceutical composition comprising acompound having the following formula

wherein Z¹ is O, S, SO₂, NH, or NR_(a), R_(a), being C₁₋₆ alkyl; X¹ isO, S, CH₂, two singly bonded H, CH(R_(b)) in the E or Z configuration,or C(R_(b)) (R_(c)) in the E or Z configuration, each of R_(b) andR_(c), independently, being C₁₋₆ alkyl, C₆₋₁₂ aryl, C₃₋₈ cycloalkyl,C₃₋₈ heteroaryl, C₃₋₈ heterocyclic radical, or halogen, X¹ being twosingly bonded H when Z¹ is SO₂; Z² is O, S, NH, NR_(d), CHR¹, or CHOR¹in the (R) or (S) configuration, wherein R_(d) is C₁₋₆ alkyl and R¹ isH, halogen, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl,NR_(d)R_(e) (except where Z² is CHOR¹), or the side chain of a naturallyoccurring α-amino acid, or R¹ and R² taken together are a bivalentmoiety, provided that when R¹ and R² are taken together, Z¹ is NH orNR_(a) and Z² is CHR¹; R_(e) being H, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆alkenyl, or C₂₋₆ alkynyl, and the bivalent moiety forming a C₃₋₈cycloalkyl, C₃₋₈ heteroaryl, C₃₋₈ heterocyclic radical, or C₆₋₁₂ aryl,where the H in CHR¹ is deleted when R₁ and R₂ taken together form a C₃₋₈heteroaryl or C₆₋₁₂ aryl; R² is C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆alkenyl, azido, C₂₋₆ alkynyl, halogen, OR_(f) SR_(f), NR_(f)R_(g),—ONR_(f)R_(g), —NR_(g)(OR_(f)), or —NR_(g)(SR_(f)) (each of R_(f) andR_(g), independently, being H, C₁₋₆, alkyl, C₁₋₆ haloalkyl, C₂₋₆alkenyl, or C₂₋₆ alkynyl), or R¹ and R² taken together are a bivalentmoiety, the bivalent moiety forming a C₃₋₈cycloalkyl, C₃₋₈ heteroaryl,C₃₋₈ heterocyclic radical, or C₆₋₁₂ aryl, where the H in CHR¹ is deletedwhen R₁ and R₂ taken together form a C₃₋₈ heteroaryl or C₆₋₁₂ aryl; A¹is H, the side chain of any naturally occurring α-amino acid, or is ofthe following formula, —(CH₂)_(m)—Y—(CH₂)_(n)—R³X³ wherein Y is O, S,C═O, C═S, —(CH═CH)—, vinylidene, —C═NOR_(h), —C═NNR_(i)R_(i,), sulfonyl,methylene, CHX⁴ in the (R) or (S) configuration, or deleted, X⁴ beinghalogen, methyl, halomethyl, OR_(h), SR_(h), NR_(i)R_(i′),—NR_(i)(OR_(h)), or —NR_(i)(NR_(i)R_(i′)), wherein R_(h) is selectedfrom H, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, and C₂₋₆ alkynyl,C₁₋₁₀ acyl, C₁₋₆ alkylsulfonyl, and C₆₋₁₀ arylsulfonyl, and each ofR_(i) and R_(i′), independently is selected from H, C₁₋₆ alkyl, C₁₋₆haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, and C₁₋₁₀ acyl; m is 0, 1, 2, or3, and n is 0, 1, 2, or 3; and R³ is straight chain or branched C₁₋₈alkylidene, straight chain or branched C₁₋₈ alkylene, C₃₋₁₀cycloalkylidene, C₃₋₁₀ cycloalkylene, phenylene, C₆₋₁₄ arylalkylidene,C₆₋₁₄ arylalkylene, or deleted, and X³ is H, hydroxyl, thiol, carboxyl,amino, halogen, (C₁₋₆ alkyl)oxycarbonyl, (C₇₋₁₄ arylalkyl)oxycarbonyl,or C₆₋₁₄ aryl; or R³ and X³ taken together are the side chain of anynaturally occurring α-amino acid where the carbon to which A¹ and L⁰ areattached is a quaternary carbon; and L⁰ is —(C═X²)—L¹, —(C═X²)—L², or—C(L²)—CH₂—O, wherein X² is O or S; L² is H, C₁₋₆ haloalkyl, C₁₋₆ alkyl,or C₆₋₁₄ aryl, and L¹ is a moiety having 0 to 25 carbon atoms, 1 to 10heteroatoms, and 0 to 6 halogen atoms, L¹ being linked by an oxygen orsulfur atom to the carbon atom bonded to X²; where A¹ is C₅₋₂₀ alkylwhen L⁰ is carboxyl or (C₁₋₄ alkyl)oxycarbonyl and A¹ is alkyl; whereonly one of A¹ and L⁰ is selected from carboxyl and (C₁₋₄alkyl)oxycarbonyl; and where L¹ has between 0 and 3 non-aromatic acycliccarbon atoms when it has 5 heteroatoms; and a pharmaceuticallyacceptable carrier.
 2. A pharmaceutical composition of claim 1, whereinZ² is CHR¹ and Z¹ is NH or NR_(a).
 3. A pharmaceutical composition ofclaim 1, wherein L¹ is linked by a sulfur atom to the carbon atom bondedto X².
 4. A pharmaceutical composition of claim 3, wherein L¹ issubstituted or unsubstituted C₆₋₂₀ arylalkyl-carbonylthio, C₆₋₂₀arylthio, C₂₋₈ alkylcarbonylthio, or C₁₋₁₂ alkylthio.
 5. Apharmaceutical composition of claim 1, wherein Z¹ is NH or NR_(a); X¹ isO or S; Z² is CHR¹ or CHOR¹, R¹ being H, halogen, C1-6 alkyl, or C₁₋₆haloalkyl; R² is OR_(f); and L⁰ is —(C═X²)—L¹, X² being O and L¹ beinglinked by a sulfur atom to the carbon atom bonded to X².
 6. Apharmaceutical composition of claim 5, comprising a compound having aformula selected from


7. The pharmaceutical composition of claim 1, wherein Z¹ is NH; X¹ is O;Z² is CHR¹, R¹ being C₁₋₆ alkyl; R² is OR_(f), R_(f) being H; A¹ is—(CH₂)_(m)—Y—(CH₂)_(n)R³X³, where m and n are each 0, Y is CHX⁴, X⁴being OH, R³ is straight chain or branched C₁₋₈ alkylidene, and X³ is H;and L⁰ is —(C═X²)—L¹, X² being O and L¹ being linked by a sulfur atom tothe carbon bonded to X².
 8. The pharmaceutical composition of claim 7,comprising a compound of the formula